Hyperphotosynthetic Organisms

ABSTRACT

The present disclosure identifies pathways and mechanisms to confer improved industrial fitness on engineered organisms. It also discloses engineered organisms having improved industrial fitness. Synthetic biologic engineering modules are disclosed that provide for light capture, carbon dioxide fixation, NADH production, NADPH production, thermotolerance, pH tolerance, flue gas tolerance, salt tolerance, nutrient independence and near infrared absorbance. The disclosed engineered organisms can include one or more of these modules. Also provided are methods of using the engineered organism to produce carbon-based products of interest, biomass or pharmaceutical agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser. No. 12/268,406, filed on Nov. 10, 2008, which claims the benefit of U.S. Provisional Applications 60/987,046, filed on Nov. 10, 2007; 61/032,169 filed on Feb. 28, 2008; and 61/090,933, filed on Aug. 22, 2008. The contents of each of these priority applications are incorporated by reference herein for all purposes.

SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically as a text file named “17190US_sequencelisting.txt,” created on Aug. 20, 2010, with a size of 26,465 bytes. The sequence listing consists of 22 sequences and is incorporated by reference.

FIELD

The present invention relates to the field of synthetic biology, and more particularly to industrialized photoautotrophic organisms designed to efficiently convert carbon dioxide and light into biomass and carbon-based products of interest.

BACKGROUND

Photosynthesis is a process by which biological entities utilize sunlight and CO₂ to produce sugars for energy. Existing photoautotrophic organisms (i.e., plants, algae, and photosynthetic bacteria) are poorly suited for industrial bioprocessing. In particular, most organisms have slow doubling times (10-72 hrs) compared to industrialized heterotrophic organisms such as Escherichia coli (20 minutes). Additionally, photoautotrophic organisms are often susceptible to moderate variations in common environmental stresses including pH, temperature and salt tolerance. Such susceptibilities make industrial applications of photoautotrophs inefficient. Moreover, increasingly toxic environmental factors (for example, toxic pollutants including heavy metals, nitrogen and sulfer based industrial by-products) can further limit applications of photoautotrophs to particular industrial uses.

Desirable products which can potentially be produced in microorganisms (for example ethanol and other branched chain higher alcohols produced in engineered E. coli (Atsumi, et al. Nature (2008) vol 451:86-90)) have been found difficult to process in photoautotrophs because of incompatible or inefficient metabolic pathways of production or the complete absence of necessary cell based biocatalysts.

Thus, there is need in the art for improved photoautotrophic organisms that are better suited for industrial bioprocessing.

SUMMARY

The present invention provides methods and compositions for engineering pathways that stably utilize photosynthetic organisms by, e.g., genetically improving their light capture and carbon fixation efficiencies and by optimizing their growth properties for propagation in photobioreactors. The present invention also provides the resulting engineered “HyperPhotosynthetic” cells and organisms that enable efficient conversion of carbon dioxide and light into carbon-based products of interest.

Provided herein is an engineered photosynthetic cell comprising: at least one engineered nucleic acid selected from the group consisting of a light capture nucleic acid, a carbon dioxide fixation pathway nucleic acid, an NADH pathway nucleic acid, an NADPH pathway nucleic acid, a thermotolerance nucleic acid, a pH tolerance nucleic acid, a flue gas tolerance nucleic acid, a salt tolerance nucleic acid, a nutrient independence nucleic acid and a near infrared absorbance nucleic acid wherein said engineered cell expresses a heterologous protein encoded by said engineered nucleic acid, overexpresses an endogenous protein as a result of the presence of said engineered nucleic acid, downregulates an endogenous protein as a result of the presence of said engineered nucleic acid, or has a gene knocked-out as a result of the presence of said engineered nucleic acid.

Also provided herein is an engineered cell comprising a plurality of engineered light capture nucleic acids, carbon dioxide fixation pathway nucleic acids, NADH pathway nucleic acids, NADPH pathway nucleic acids, thermotolerance nucleic acids, pH tolerance nucleic acids, flue gas tolerance nucleic acids, salt tolerance nucleic acids, nutrient independence nucleic acids and near infrared absorbance nucleic acids.

The invention also provides an engineered photosynthetic cell comprising an engineered carbon fixation pathway nucleic acid encoding one or more proteins selected from the group consisting of 3-hydroxypropionate cycle protein, Calvin cycle protein, carbon-acetyl-CoA flux protein, gluconeogenesis pathway protein, glyoxylate shunt pathway protein, pyruvate synthesis pathway protein, reductive TCA cycle protein and Woods-Ljungdahl pathway protein.

The invention also provides an engineered photosynthetic cell comprising an engineered NADH pathway nucleic acid encoding one or more proteins selected from the group consisting of NAD⁺-dependent isocitrate dehydrogenase—idh1 protein; NAD⁺-dependent isocitrate dehydrogenase—idh2 protein; malate dehydrogenase protein; soluble pyridine nucleotide transhydrogenase (sthA or udhA) protein; membrane-bound pyridine nucleotide transhydrogenase, subunit alpha, pntA protein; membrane-bound pyridine nucleotide transhydrogenase, subunit beta, pntB protein; and NADH:ubiquinone oxidoreductase (nuo) operon protein.

The invention also provides an engineered photosynthetic cell comprising an engineered NADPH pathway nucleic acid encoding one or more proteins selected from the group consisting of a glucose-6-phosphate dehydrogenase; zwf protein; 6-phosphogluconolactonase—pgl protein; 6-phosphogluconate dehydrogenase, gnd protein; NADP-dependent isocitrate dehydrogenase protein; NADP-dependent malic enzyme; soluble pyridine nucleotide transhydrogenase (sthA or udhA) protein; membrane-bound pyridine nucleotide transhydrogenase, subunit alpha, pntA protein; and membrane-bound pyridine nucleotide transhydrogenase, subunit beta, pntB protein.

The invention also provides an engineered photosynthetic cell comprising an engineered thermotolerance nucleic acid encoding one or more proteins selected from the group consisting of betaine biosynthesis protein; photosystem stability and repair protein; and protein folding protein.

The invention also provides an engineered photosynthetic cell comprising an engineered pH tolerance nucleic acid encoding one or more proteins selected from the group consisting of glutamate decarboxylase A (GadA) protein; glutamate decarboxylase beta (GadB) protein; glutamate:gamma-aminobutyric acid antiporter (GadC) protein; biodegradative arginine decarboxylase (AdiA) protein; arginine:agmatin antiporter (AdiC) protein; chaperone protein dnaK2 (DnaK) protein; DNA-directed RNA polymerase, sigma subunit (sll0306) protein; Zn-dependent protease (sll0528) protein; metal-dependent phosphoesterase (sll0549) protein; acid-stress related membrane protein (sll0939) protein; heat shock molecular chaperone (sll1514) protein; mannose-1-phosphate guanyltransferase (sll1558) protein; RNA polymerase sigma factor (sll2012) protein; carboxyl-terminal processing protease (slr0008) protein; molecular chaperone (slr0093) protein; geranylgeranyl pyrophosphate synthase (slr0611) protein; CheY-like receiver (slr1214) protein; signal transduction histidine kinase (slr1285) protein; superoxide dismutase (slr1516) protein; hydrogenase expression/formation protein (slr1675) protein; esterase (slr1916) protein; hydrogenase component protein (ssl3044) protein; chloride channel protein; and acid-stress tolerance protein.

In addition, the invention further provides an engineered photosynthetic cell comprising an engineered flue gas tolerance nucleic acid encoding one or more proteins selected from the group consisting of NO_(x) tolerance protein, SO_(x) tolerance protein, mercury tolerance protein, metal tolerance protein and particulate aerosol tolerance protein.

In yet another embodiment, the invention provides an engineered photosynthetic cell comprising an engineered salt tolerance nucleic acid encoding one or more proteins selected from the group comprising Na⁺/H⁺ antiporter protein (apnhaP), catalase protein (katG), salt and cadmium stress related protein (CW80Cd404), breast basic conserved protein (bbc1) and betaine biosynthesis protein.

In still another embodiment, the invention provides an engineered photosynthetic cell comprising an engineered near infrared absorbance nucleic acid, wherein the nucleic acid comprises a chlorophyll d biosynthetic nucleic acid.

In still another embodiment, the invention provides an engineered photosynthetic cell comprising an engineered nutrient independence nucleic acid encoding one or more a proteins selected from the group consisting of an ammonia assimilation protein, a nitrate/nitrite assimilation protein, a nitrate/nitrite assimilation-nitrite tolerance protein, a nitrogen fixation protein, an urea assimilation protein, a Vitamin B₁₂ biosynthesis protein, a Vitamin B₁₂ biosynthesis—Co²⁺ transport protein and a Vitamin B₁₂ independence protein. In certain related embodiments, the Vitamin B₁₂ biosynthesis protein comprises one or more proteins selected from the group consisting of uroporphyrin-III C-methyltransferase cobaltochelatase (cysG) protein; sirohydrochlorin cobaltochelatase (cbiK) protein; precorrin-2 C20-methyltransferase (cbiL) protein; precorrin-3B methylase (cbiH) protein; bifunctional CbiG/precorrin methyltransferase (cbiG) protein; precorrin-4 C11-methyltransferase (cbiF) protein; cobalamin biosynthesis protein (cbiD); NADPH-dependent precorrin-6A reductase (cbiJ) protein; precorrin-6B C5,15-methyltransferase (cbiE) protein; precorrin-6B C12 decarboxylase (cbiT) protein; precorrin-8X-methylmutase (cbiC) protein; cobyrinic acid A,C-diamide synthase (cbiA) protein; cob(I)yrinic acid a,c-diamide adenosyltransferase (btuR) protein; cobyrinic acid synthase (cbiP) protein; cobyric acid decarboxylase (cobD) protein; adenosylcobinamide-phosphate synthase (cbiB) protein; alpha ribazole-5′-P phosphatase (cobC) protein; cobalamin(5′-phosphate) synthase (cobS) protein; cobinamide phosphate guanylyl transferase (cobU) protein; and nicotinate-nucleotide dimethylbenzimidazole-P phosphoribosyl transferase (cobT) protein. In yet another related embodiment, the Vitamin B₁₂ biosynthesis—Co²⁺ transport protein comprises one or more proteins selected from the group consisting of ABC-type Co²⁺ transport system, permease component protein; ABC-type cobalt transport system, periplasmic component protein; and ABC-type cobalt transport system, permease component protein. In other related embodiments, the Vitamin B₁₂ independence protein comprises one or more proteins selected from the group consisting of 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (metE) protein; ribonucleoside-diphosphate reductase, alpha subunit (nrdA) protein; and ribonucleoside-diphosphate reductase, beta subunit (nrdB) protein. In a related embodiment, the metE protein is from Escherichia coli K12 (accession number NP_(—)418273.1 (SEQ ID NO: 20)) or Thermosynechococcus elongatus BP-1 (accession number NP_(—)681881 (SEQ ID NO: 21)).

In various embodiments of the invention, the engineered nucleic acid encodes a heterologous protein that is expressed by the engineered cell, causes overexpression of an endogenous protein within the engineered cell, causes downregulation of an endogenous protein in the engineered cell, or causes a gene knock-out in the engineered cell.

In another embodiment of the invention, the engineered cell is used to produce biomass, carbon-based products of interest or pharmaceutical agents.

In yet other embodiments, the invention provides an engineered carbon-fixing cell comprising at least one engineered nucleic acid selected from the group consisting of thermotolerance nucleic acid, pH tolerance nucleic acid, flue gas tolerance nucleic acid, salt tolerance nucleic acid, nutrient independence nucleic acid and near infrared absorbance nucleic acid, wherein said engineered cell expresses heterologous protein encoded by said engineered nucleic acid, overexpresses endogenous protein as a result of the presence of said engineered nucleic acid, downregulates endogenous protein as a result of the presence of said engineered nucleic acid, or has a gene knocked-out as a result of the presence of said engineered nucleic acid is provided.

In certain embodiments of the invention, the engineered cell is an autotrophic cell. In certain other embodiments, the engineered cell is a photoautotrophic cell. In various embodiments of the invention, the engineered cell is selected from a group consisting of plant, prokaryote, eukaryote, yeast, filamentous fungi, protozoa, algae and synthetic cells.

Also disclosed is a method to produce carbon-based products of interest, comprising the steps of: a) engineering a cell to express at least one engineered nucleic acid selected from the group consisting of light capture nucleic acid, carbon dioxide fixation pathway nucleic acid, NADH pathway nucleic acid, NADPH pathway nucleic acid, thermotolerance nucleic acid, pH tolerance nucleic acid, flue gas tolerance nucleic acid, salt tolerance nucleic acid, nutrient independence nucleic acid and near infrared absorbance nucleic acid; b) said cell expressing a heterologous protein encoded by said engineered nucleic acid, overexpressing endogenous protein as a result of the presence of said engineered nucleic acid, downregulating endogenous protein as a result of the presence of said engineered nucleic acid, or having a gene knocked-out as a result of the presence of said engineered nucleic acid; and c) culturing said cell in the presence of CO₂ and light to produce the carbon-based products of interest.

FIGURES

FIG. 1 provides Table 1 which identifies genes that can be expressed or upregulated in association with the engineering of various modules of the invention.

FIG. 2 provides Table 2 which identifies genes that can be downregulated or knocked out in association with the engineering of various modules of the invention.

FIG. 3 depicts viability of wild-type and genetically engineered Synechococcus sp. PCC 7002 on A⁺ media agarose plates without B12.

FIG. 4 provides synthetic schemes for chlorophyll d (Chl d) derivation from chlorophyll a (Chl a) with Chlorophyllide, a precursor of Chl, shown here for simplicity.

FIG. 5 provides a query sequence (SEQ ID NO: 19) for an epoxide hydrolase search.

FIG. 6 provides Table 3 which lists genes identified as those encoding oxygenases in the A. marina genome.

FIG. 7 provides Table 4 which lists genes identified as those encoding epoxide reductases in the A. marina genome.

FIG. 8 provides Table 5 which lists genes identified as those encoding SAM-utilizing oxygenases in the A. marina genome.

FIG. 9 provides Table 6 which lists genes identified as those encoding photosystem proteins in the A. marina genome.

FIG. 10A is a photograph of an agarose gel showing PCR confirmation of transgenic Synechococcus sp. PCC7002 strains: JCC1-UdhA (lanes 2 and 3); JCC1-ZWF (lane 4); and JCC1-SAR86 (lane 5). FIG. 10B is a photograph of an agarose gel showing PCR products from JCC136.

FIG. 11 depicts a graph of ethanol production (left) and ethanol to OD ratio (right) as a function of time in Synechococcus sp. PCC7002 strains JCC136 and JCC136-FbpI.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for amino acid residues, as defined in 37 C.F.R. 1.822. In the accompanying amino acid sequence listing:

ABBREVIATIONS AND TERMS

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. For example, reference to “comprising a cell” includes one or a plurality of such cells, and reference to “comprising a thioesterase” includes reference to one or more thioesterase peptides and equivalents thereof known to those of ordinary skill in the art, and so forth. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

Accession Numbers: The accession numbers throughout this description correspond to those found in the following databases: NCBI (National Center for Biotechnology Information), TIGR (The Institute for Genomic Research), and KEGG (Kyoto Encyclopedia of Genes and Genomes). The accession numbers are as provided in the databases on Nov. 10, 2007.

Enzyme Classification Numbers (EC): The EC numbers provided throughout this description are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. The EC numbers are as provided in the database on Nov. 10, 2007.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.

Codon: Triplets of nucleotides, referred to as codons, in DNA molecules code for amino acids in a peptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Endogenous: As used herein with reference to a nucleic acid molecule and a particular cell or microorganism refers to a nucleic acid sequence or peptide that is in the cell and was not introduced into the cell using recombinant engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature is considered to be endogenous. A gene is still considered endogenous if the control sequences, such as a promoter or enhancer sequences that activate transcription or translation have been altered through recombinant techniques.

Exogenous: As used herein with reference to a nucleic acid molecule and a particular cell or microorganism refers to a nucleic acid sequence or peptide that was not present in the cell when the cell was originally isolated from nature. For example, a nucleic acid that originated in a different microorganism and was engineered into an alternate cell using recombinant DNA techniques or other methods for delivering said nucleic acid is considered to be exogenous.

Expression: The process by which a gene's coded information is converted into the molecules that support the structures and functions of a cell, such as a protein, transfer RNA, or ribosomal RNA. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (for example, transfer and ribosomal RNAs).

Overexpression: Overexpression refers to any state in which a gene is caused to be transcribed at an elevated rate as compared to the endogenous transcription rate for that gene. In some examples, overexpression additionally includes an elevated rate of translation of the gene compared to the endogenous translation rate for that gene. Methods of testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels can be assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Furthermore, a gene is considered to be overexpressed when it exhibits elevated activity compared to its endogenous activity, which may occur, for example, through reduction in concentration or activity of its inhibitor, or via expression of mutant version with elevated activity. In preferred embodiments, when the host cell encodes an endogenous gene with a desired biochemical activity, it is useful to overexpress an exogenous gene, which allows for more explicit regulatory control in the bioprocessing and a means to potentially mitigate the effects of central metabolism regulation, which is focused around the native genes explicitly.

Downregulation: Downregulation refers to any state in which a gene is caused to be transcribed at a reduced rate compared to the endogenous gene transcription rate for that gene. In certain embodiments, gene expression is downregulated via expression of nucleic acids, such as antisense oligonucleotides, double-stranded RNA, small interfering RNA, small hairpin RNA, microRNAs, ribozymes, and the like. In some examples, downregulation additionally includes a reduced level of translation of the gene compared to the endogenous translation rate for that gene. Furthermore, a gene is considered to be downregulated when it exhibits decreased activity compared to its endogenous activity, which may occur, for example, through an increase in concentration or activity of its inhibitor, or via expression of mutant version with reduced activity. Methods of testing for downregulation are well known to those in the art, for example the transcribed RNA levels can be assessed using RT-PCR and proteins levels can be assessed using SDS-PAGE analysis.

Knock-out: A gene whose level of expression or activity has been reduced to zero. In some examples, a gene is knocked-out via deletion or replacement of some or all of its coding sequence. In other examples, a gene is knocked-out via introduction or removal of one or more nucleotides into its open-reading frame, which results in translation of a non-sense or otherwise non-functional protein product.

Autotroph: Autotrophs (or autotrophic organisms) are organisms that produce complex organic compounds from simple inorganic molecules and an external source of energy, such as light (photoautotroph) or chemical reactions of inorganic compounds.

Heterotroph: Heterotrophs (or heterotrophic organisms) are organisms that, unlike autotrophs, cannot derive energy directly from light or from inorganic chemicals, and so must feed on organic carbon substrates. They obtain chemical energy by breaking down the organic molecules they consume. Heterotrophs include animals, fungi, and numerous types of bacteria.

Hyperphotosynthetic: a cell or organism expressing photosynthetic proteins that through recombinant DNA techniques has been specifically engineered to express endogenous and/or exogenous nucleic acids that result in one or more functional improvements related to industrial bioprocessing and the conversion of carbon dioxide and light into reduced carbon products or cell mass. A Hyperphotosynthetic cell also encompasses a cell or organism engineered as described above that has been evolved or mutagenized to achieve one or more functional improvements.

Hydrocarbon: generally refers to a chemical compound that consists of the elements carbon (C), optionally oxygen (O), and hydrogen (H).

Biosynthetic pathway: Also referred to as “metabolic pathway,” refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another. For example, a hydrocarbon biosynthetic pathway refers to the set of biochemical reactions that convert inputs and/or metabolites to hydrocarbon product-like intermediates and then to hydrocarbons or hydrocarbon products. Anabolic pathways involve constructing a larger molecule from smaller molecules, a process requiring energy. Catabolic pathways involve breaking down of larger: molecules, often releasing energy.

Cellulose: Cellulose [(C₆H₁₀O₅)_(n)] is a long-chain polymer polysaccharide carbohydrate, of beta-glucose. It forms the primary structural component of plants and is not digestible by humans. Cellulose is a common material in plant cell walls and was first noted as such in 1838. It occurs naturally in almost pure form only in cotton fiber; in combination with lignin and any hemicellulose, it is found in all plant material.

Surfactants: Surfactants are substances capable of reducing the surface tension of a liquid in which they are dissolved. They are typically composed of a water-soluble head and a hydrocarbon chain or tail. The water soluble group is hydrophilic and can be either ionic or nonionic, and the hydrocarbon chain is hydrophobic.

Biofuel: A biofuel is any fuel that derives from a biological source.

Engineered nucleic acid: An “engineered nucleic acid” is a nucleic acid molecule that includes at least one difference from a naturally-occurring nucleic acid molecule. An engineered nucleic acid includes all exogenous modified and unmodified heterologous sequences (i.e., sequences derived from an organism or cell other than that harboring the engineered nucleic acid) as well as endogenous genes, operons, coding sequences, or non-coding sequences, that have been modified, mutated, or that include deletions or insertions as compared to a naturally-occurring sequence. Engineered nucleic acids also include all sequences, regardless of origin, that are linked to an inducible promoter or to another control sequence with which they are not naturally associated. Engineered nucleic acids further include all sequences that can be used to down-regulate or knock out expression of an endogenous gene. These include anti-sense molecules, RNAi molecules, constructs for producing homologous recombination, cre-lox constructs, and the like.

Light capture nucleic acid: A “light capture nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes one or more proteins that convert light energy (i.e., photons) into chemical energy such as a proton gradient, reducing power, or a molecule containing at least one high-energy phosphate bond such as ATP or GTP. Exemplary light capture nucleic acids include those encoding light-activated proton pumps such as rhodopsin, xanthorhodopsin, proteorhodopsin and bacteriorhodopsin, as well as nucleic acids encoding proteins that directly (light harvesting complexes, LHC I and LHC II) or indirectly (truncated light harvesting antenna, tla1) modulate light harvesting capture efficiencies. A light capture nucleic acids further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression reduces light capture.

Carbon dioxide fixation pathway nucleic acid: A “carbon dioxide fixation pathway nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein that enables autotrophic carbon fixation. A carbon dioxide fixation pathway nucleic acid also includes nucleic acids that direct carbon flux to key cellular intermediates required for efficient growth or carbon-based product formation, such as acetyl-CoA. Exemplary carbon dioxide fixation pathway nucleic acids includes those encoding propionyl-CoA carboxylase, pyruvate synthase, formate dehydrogenase, and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). A carbon dioxide fixation pathway nucleic acids further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression reduces carbon dioxide fixation.

NADH pathway nucleic acid: A “NADH pathway nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein to maintain an appropriately balanced supply of reduced NAD for carrying out carbon fixation. Exemplary NADH pathway nucleic acids include those encoding an NAD⁺-dependent isocitrate dehydrogenase and malate dehydrogenase. A NADH pathway nucleic acid further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression detracts from maintaining an appropriately balanced supply of reduced NAD for carrying out carbon fixation and other necessary biological processes.

NADPH pathway nucleic acid: A “NADPH pathway nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein to maintain an appropriately balanced supply of reduced NADPH for carrying out carbon fixation. Exemplary NADPH pathway nucleic acids include those encoding phosphogluconolactonase and soluble pyridine nucleotide transhydrogenase. A NADPH pathway nucleic acid further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression detracts from maintaining an appropriately balanced supply of reduced NADP for carrying out carbon fixation and other necessary biological processes.

Thermotolerance nucleic acid: A “thermotolerance nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition results in an increase in thermotolerance. Exemplary thermotolerance nucleic acids include those encoding ClpC/Hsp100, groESL1, HspA, and PsbU. A thermotolerance nucleic acid further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression impairs thermotolerance

pH tolerance nucleic acid: A “pH tolerance nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition enables growth at an elevated or reduced pH. Exemplary pH tolerance nucleic acids include those encoding glutamate decarboxylase and superoxide dismutase. A pH tolerance nucleic acid further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression impairs pH tolerance.

Flue gas tolerance: A “Flue gas tolerance nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition enables growth in the presence flue gas components including carbon dioxide, SO_(x), NO_(x), and N₂. Exemplary flue gas tolerance nucleic acids include those encoding superoxide dismutase, catalase, cysteine synthase, and NirK. A flue gas tolerance nucleic acid further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression impairs flue gas tolerance.

Nutrient independence nucleic acid: A “nutrient independence nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition enables propagation in the absence of, or under reduced concentrations of, an exogenous nutrient. Exemplary nutrient independence nucleic acids include those encoding MetE and NrdB. A nutrient independence gene further includes a nucleic acid used to reduce the expression of or knock out one or more endogenous genes whose expression impairs propagation in the absence of, or under reduced concentrations of, an exogenous nutrient.

Salt-tolerance nucleic acid: A “salt tolerance nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition enables propagation under conditions of elevated salinity, such as sea water Exemplary salt tolerance nucleic acids include those encoding Na+/H+ antiporter, and breast basic conserved.

Acetyl-CoA flux nucleic acid: An “acetyl-CoA flux nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition results in an increase in acetyl-CoA produced over a unit of time. Example nucleic acids that may be overexpressed include pantothenate kinase and pyruvate dehydrogenase. Nucleic acids that may be downregulated, inhibited, or knocked-out include acyl coenzyme A dehydrogenase, biosynthetic glycerol 3-phosphate dehydrogenase, and lactate dehydrogenase.

Chlorophyll d nucleic acid: A “chlorophyll d nucleic acid” refers to a nucleic acid that alone or in combination with another nucleic acid encodes a protein whose overexpression, downregulation, or inhibition results in the biosynthesis of chlorophyll d and its incorporation in reaction centers, photosystem (PS)I and PSII. A chlorophyll d nucleic acid enables propagation under, e.g., conditions in which the photosynthetically available radiation is likely completely used by organisms that absorb light using chlorophyll a and/or chlorophyll b such that the environment has low visible light intensity but high near infrared intensity where no other photosynthetic organisms absorb strongly so that an organism that uses chlorophyll d for light capture can thrive. Alternatively, a chlorophyll d nucleic acid enables propagation under conditions in which the wavelength of artificial light used for illumination is selected to allow propagation of an organism that uses chlorophyll d for light capture.

Sequence identity: The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

Comparison window: A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

DETAILED DESCRIPTION OF THE INVENTION Organisms

The instant invention enables conversion of a photoautotrophic organism into a Hyperphotosynthetic organism. Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria.

Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix Simmondsia, and Zea.

Algae and cyanobacteria include but are not limited to the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.

Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium.

Green sulfur bacteria include but are not limited to the following genera: Chlorobium, Clathrochloris, and Prosthecochloris,

Purple sulfur bacteria include but are not limited to the following genera: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis,

Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.

HyperPhotosynthetic conversion requires extensive genetic modification (Tables 1 and 2); thus, in preferred embodiments the parental photoautotrophic organism can be transformed with exogenous DNA.

Preferred organisms for HyperPhotosynthetic conversion include: Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zea mays (plants), Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae), Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidum and Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter capsulatus, and Rhodopseudomonas palusris (purple non-sulfur bacteria).

Propagation

Methods for cultivation of photosynthetic organisms in liquid media and on agarose-containing plates are well known to those skilled in the art (see, e.g., websites associated with ATCC, and with the Institute Pasteur). For example, Synechococcus sp. PCC 7002 cells (available from the Pasteur Culture Collection of Cyanobacteria) are cultured in BG-11 medium (17.65 mM NaNO₃, 0.18 mM K₂HPO₄, 0.3 mM MgSO₄, 0.25 mM CaCl₂, 0.03 mM citric acid, 0.03 mM ferric ammonium citrate, 0.003 mM EDTA, 0.19 mM Na₂CO₃, 2.86 mg/L H₃BO₃, 1.81 mg/L MnCl₂, 0.222 mg/L ZnSO₄, 0.390 mg/L Na₂MoO₄, 0.079 mg/L CuSO₄, and 0.049 mg/L Co(NO₃)₂, pH 7.4) supplemented with 16 μg/L biotin, 20 mM MgSO₄, 8 mM KCl, and 300 mM NaCl (see, e.g., website associated with the Institute Pasteur, and Price G D, Woodger F J, Badger M R, Howitt S M, Tucker L. “Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl. Acad. Sci USA (2004). 101(52):18228-33). Typically, cultures are maintained at 28° C. and bubbled continuously with 5% CO₂ under a light intensity of 120 μmol photons/m²/s.

Thermosynechococcus elongatus BP-1 (available from the Kazusa DNA Research Institute, Japan) is propagated in BG11 medium supplemented with 20 mM TES-KOH (pH 8.2) as described [Iwai M, Katoh H, Katayama M, Ikeuchi M. “Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1.” Plant Cell Physiol (2004). 45(2):171-175)]. Typically, cultures are maintained at 50° C. and bubbled continuously with 5% CO₂ under a light intensity of 38 μmol photons/m²/s.

Chlamydomonas reinhardtii (available from the Chlamydomonas Center culture collection maintained by Duke University, Durham, N.C.) are grown in minimal salt medium consisting of 143 mg/L K₂HPO₄, 73 mg/L KH₂PO₄, 400 mg/L NH₄NO₃, 100 mg/L MgSO₄-7H2O, 50 mg/L CaCl₂-2 H20, 1 mL/L trace elements stock, and 10 mL/L 2.0 M MOPS titrated with Tris base to pH 7.6 as described (Geraghty A M, Anderson J C, Spalding M H. “A 36 kilodalton limiting-CO2 induced polypeptide of Chlamydomonas is distinct from the 37 kilodalton periplasmic anhydrase.” Plant Physiol (1990). 93:116-121). Typically, cultures are maintained at 24° C. and bubbled with 5% CO₂ in air, under a light intensity of 60 μmol photons/m²/s.

The above define typical propagation conditions. As appropriate, incubations are performed using alternate media or gas compositions, alternate temperatures (5-75° C.), and/or light fluxes (0-5500 μmol photons/m²/s).

Light is delivered through a variety of mechanisms, including natural illumination (sunlight), standard incandescent, fluorescent, or halogen bulbs, or via propagation in specially-designed illuminated growth chambers (for example Model LI15 Illuminated Growth Chamber (Sheldon Manufacturing, Inc. Cornelius, Oreg.). For experiments requiring specific wavelengths and/or intensities, light is distributed via light emitting diodes (LEDs), in which wavelength spectra and intensity can be carefully controlled (Philips).

Carbon dioxide is supplied via inclusion of solid media supplements (i.e., sodium bicarbonate) or as a gas via its distribution into the growth incubator or media. Most experiments are performed using concentrated carbon dioxide gas, at concentrations between 1 and 30%, which is directly bubbled into the growth media at velocities sufficient to provide mixing for the organisms. When concentrated carbon dioxide gas is utilized, the gas originates in pure form from commercially-available cylinders, or preferentially from concentrated sources including off-gas or flue gas from coal plants, refineries, cement production facilities, natural gas facilities, breweries, and the like.

Plasmids

Plasmids relevant to genetic engineering typically include at least two functional elements 1) an origin of replication enabling propagation of the DNA sequence in the host organism, and 2) a selective marker (for example an antibiotic resistance marker conferring resistance to ampicillin, kanamycin, zeocin, chloramphenicol, tetracycline, spectinomycin, and the like). Plasmids are often referred to as “cloning vectors” when their primary purpose is to enable propagation of a desired heterologous DNA insert. Plasmids can also include cis-acting regulatory sequences to direct transcription and translation of heterologous DNA inserts (for example, promoters, transcription terminators, ribosome binding sites); such plasmids are frequently referred to as “expression vectors.” When plasmids contain functional elements that allow for propagation in more than one species, such plasmids are referred to as “shuttle vectors.” Shuttle vectors are well known to those in the art. For example, pSE4 is a shuttle vector that allows propagation in E. coli and Synechococcus [Maeda S, Kawaguchi Y, Ohy T, and Omata T. J. Bacteriol. (1998). 180:4080-4088]. Shuttle vectors are particularly useful in the present invention to allow for facile manipulation of genes and regulatory sequences in E. coli prior to transformation into the photoautotrophic organism of interest.

Transformation Techniques

Standard methods for transformation of prokaryotes are well known to those skilled in the art [Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.; Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.; and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (through and including the 1997 Supplement)].

Many prokaryotic organisms are naturally competent; others can be rendered competent by chemical treatments. Non-limiting examples of transformation techniques include direct incubation in the presence of exogenous DNA, transformation by heat-shock, transformation by electro-poration, transformation by biolistic particle bombardment, transformation via addition of lipids or fusogenic agents (i.e., polyethylene glycol), conjugation with a heterologous microorganism, or transduction via viral particles.

Synechococcus sp. PCC 7002 cells are transformed according to the optimized protocol previously described [Essich E S, Stevens Jr E, Porter R D “Chromosomal Transformation in the Cyanobacterium Agmenellum quadruplicatum”. J Bacteriol (1990). 172(4):1916-1922]. Cells are grown in Medium A (18 g/L NaCl, 5 g/L MgSO₄. 7 H₂0, 30 mg/L Na₂EDTA, 600 mg/L KCl, 370 mg/L CaCl₂.2 H₂O, 1 g/L NaNO₃, 50 mg/L KH₂PO₄, 1 g/L Trizma base pH 8.2, 4 μg/L Vitamin B12, 3.89 mg/L FeCl₃.6 H₂0, 34.3 mg/L H₃BO₃, 4.3 mg/L MnCl₂.4 H₂0, 315 μg/L ZnCl₂, 30 μg/L MoO₃, 3 μg/L CuSO₄.5 H₂0, 12.2 μg/L CoCl₂.6 H₂0) [Stevens S E, Patterson C O P, and Myers J. “The production of hydrogen peroxide by green algae: a survey.” J. Phycology (1973). 9:427-430] plus 5 g/L of NaNO₃ to approximately 10⁸ cells/mL. Nine volumes of cells are mixed with 1 volume of 1-10 μg/mL DNA in 0.15 M NaCl/0.015 M Na₃citrate and incubated at 27-30° C. for 3 hours before addition of 1 volume of DNaseI to a final concentration of 10 μg/mL. The cells are plated in 2.5 mL of 0.6% medium A overlay agar that was tempered at 45° C. and incubated. Cells are challenged with antibiotic by under-laying 2.0 mL of 0.6% medium A agar containing appropriate concentration of antibiotic with a sterile Pasteur pipette. Transformants are picked 3-4 days later. Selections are typically performed using 200 μg/ml kanamycin, 8 μg/ml chloramphenicol, 10 μg/ml spectinomycin on solid media, whereas 150 μg/ml kanamycin, 7 μg/ml chloramphenicol, and 5 μg/ml spectinomycin are employed in liquid media.

Thermosynechococcus elongatus BP-1 cells are transformed according to the optimized protocol previously described [Iwai M, Katoh H, Katayama M, and Ikeuchi M. “Improved genetic transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. Plant Cell Physiol (2004). 45(2):171-175]. Mid-exponential phase cultures are incubated with exogenous DNA (typically 3-20 μg) and electroporated at a field strength of 10 kV/cm using a BioRad Gene Pulsur Xcell (Bio-Rad Laboratories, Hercules, Calif.). Following electroporation, the cells are recovered in 1 ml BG11 medium for 24-hrs at 45° C. Cells are plated directly on BG11 plates containing the appropriate antibiotic or optionally pre-mixed with BG11 medium containing 0.35% (w/v) melted agar (Difco, USA).

Transformation typically involves incubation of recipient cells with purified plasmid DNA isolated from E. coli. In contrast, bacterial conjugation provides an alternate means to directly transfer DNA from one bacterial species to another. For example, techniques enabling conjugation between E. coli and cyanobacteria including Synechocystic PCC 6803 and PCC 6714 and Synechococcus PCC 7942 and PCC 6301, as well as thermophilic Synechococcus elongatus have been described [Marraccini P, Bulteau S, Cassier-Chauvat C, Mermet-Bouvier P, and Chauvat F. “A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis.” Plant Molecular Biology (1993). 23(4):905-909; Muhlenhoff U and Chauvat; “Gene transfer and manipulation in the thermophilic cyanobacterium Synechococcus elongatus.” Molecular and General Genetics MGG (1996). 252(1-2):93-100

Techniques related to the transformation of eukaryotic photoautotrophs are known to those skilled in the art. Such methods have been described in textbooks related to molecular biology (see Packer & Glaser, 1988, “Cyanobacteria”, Meth. Enzymol., Vol. 167; Weissbach & Weissbach, 1988, “Methods for plant molecular biology,” Academic Press, New York, Sambrook, Fritsch & Maniatis, 1989, “Molecular Cloning: A laboratory manual,” 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Clark M S, 1997, Plant Molecular Biology, Springer, N.Y.) and in “Methods and tools for transformation of eukaryotic algae” U.S. Pat. No. 6,027,900.

A variety of approaches can be employed for transformation of Chlamydomonas reinhardtii cells. Transformation of the Chlamydomonas reinhardtii nuclear compartment is performed as described [Kindle K L. “High-frequency nuclear transformation of Chlamydomonas reinhardtii.” Proc Natl Acad Sci (1990). 87:1228-1232]. In brief, cells are grown to a concentration of 1.5×10⁶ per ml, pelleted, and resuspended in 5% (v/v) polyethylene glycol (PEG, M_(r) 6000) from Sigma-Aldrich (St. Louis, Mo.). Subsequently, the resuspended cells are incubated with linear or circular plasmid DNA and agitated in the presence of 300 mg of 0.5-mm glass beads for 10-30 seconds at maximum speed using a Genie Vortex II mixer (Thermo Fisher Scientific, Pittsburgh, Pa.). Cells are immediately plated on agarose plates containing the appropriate antibiotic or nutrient selection. Alternately, Chlamydomonas reinhardtii cells are plated directly onto agarose plates and bombarded with 500 μg of tungsten microprojectiles coated with 1 μg of plasmid DNA using the Bio-Rad PDS-1000He particle-delivery system (Bio-Rad Laboratories; Hercules, Calif.), as previously described [Sodeinde O A and Kindle K L. “Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii.” Proc Natl Acad Sci (1993). 90:9199-9203].

Chlamydomonas reinhardtii chloroplasts are transformed as described (Kindle K L, Richards K L, Stern D B. “Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci. (2001). 88:1721-1725). In brief, cells are grown to mid log phase, optionally in the presence of 0.5 mM 5-fluorodeoxyuridine, and agitated with single or double-stranded plasmid DNA in the presence of 300 mg of 0.5-mm glass beads for 15-30 seconds at maximum speed using a Genie Vortex II mixer. Following agitation, cells are immediately plated on selective agar plates containing the appropriate concentration of antibiotics. Typically, 100 μg/ml of spectinomycin is used.

Chlamydomonas reinhardtii mitochondria are transformed as described (Remade C, Cardol P, Coosemans N, Galsne M, and Bonnefoy N. “High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes.” Proc Natl Acad Sci (2006). 103: 4771-4776.] Briefly, cells are grown in liquid Tris Acetate Phosphate media (TAP) [http://openwetware.org/wiki/Media_formula] to a concentration of 2-3×10⁶ cells/ml and 10⁸ cells are spread onto TAP plates and bombarded with tungsten beads coated with DNA using the Bio-Rad PDS-1000He apparatus under 1100 psi pressure and a partial chamber vacuum of at least 29 inches Hg. Plates are positioned approximately 7 cm from the macrocarrier assembly, optionally employing stopping screens to increase transformation efficiencies.

Tables 1 and 2 define preferred genes to convey HyperPhotosynthetic properties to an existing photoautotrophic organism.

Table 1 lists genes which are overexpressed to enhance carbon fixation rates, thermotolerance, pH tolerance, flue gas tolerance, salt tolerance, light harvesting efficiencies, reducing power generation, and nutrient independence, together with information on associated pathways, Enzyme Commission (EC) Numbers, exemplary gene names, source organism, GenBank accession numbers, and homologs from alternate sources. When the parental organism encodes a gene with the indicated enzymatic activity, it is nevertheless useful to overexpress these components to improve CO₂ fixation. In one embodiment, the native enzyme sequence is overexpressed. In preferred embodiments, it is useful to overexpress an exogenous gene, which allows for more explicit regulatory control in the bioprocess and a means to potentially mitigate the effects of central metabolism regulation, which is focused around the native genes explicitly.

The nucleotide sequences for the indicated genes (or DNA sequences that encode the identical or homologous polypeptides, but encompassing nucleotide substitutions to 1) alter expression levels based on the host organism's codon usage table; 2) add or remove secondary structure; 3) add or remove restriction endonuclease recognition sequences; and/or 4) facilitate gene synthesis and assembly) are assembled by Codon Devices Inc (Cambridge, Mass.). Alternate providers including DNA2.0 (Menlo Park, Calif.), Blue Heron Biotechnology (Bothell, Wash.), and Geneart (Regensburg, Germany), are used as noted. Sequences untenable by commercial sources may be prepared using polymerase chain reaction (PCR) from DNA or cDNA samples, or cDNA/BAC libraries. Inserts are initially propagated and sequenced in a cloning vector, such as pUC19. Importantly, primary synthesis and sequence verification of each gene of interest in pUC19 provides flexibility to transfer each unit in various combinations to alternate destination vectors to drive transcription and translation of the desired enzymes. Specific and/or unique cloning sites are included at the 5′ and 3′ ends of the open reading frames (ORFs) to facilitate molecular transfers.

The required metabolic pathways are initially encoded in expression cassettes driven by constitutive promoters which are always “on.” Many such promoters are known, for example the spc ribosomal protein operon (P_(spc)), the beta-lactamase gene promoter of pBR322 (P_(bla)), the bacteriophage lambda P_(L) promoter, the replication control promoters of plasmid pBR322 (P_(RNAI) or P_(RNAII)), or the P1 or P2 promoters of the rrnB ribosomal RNA operon [Liang S T, Bipatnath M, Xu Y C, Chen S L, Dennis P, Ehrenber M, Bremer H. Activities of Constitutive Promoters in Escherichia coli. J. Mol Biol (1999). Vol 292, Number 1, pgs 19-37], the Chlorella virus promoters described in U.S. Pat. No. 5,846,744 [“Chlorella virus promoters”], the cauliflower mosaic virus 35S promoter [Zheng X, Deng W, Luo K, Duan H, Chen Y, McAvoy R, Song S, Pei Y, Li Y. “The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters.” Plant Cell Rep (2007). 26(8):1195-1203], the constitutive petH promoter of Anabaena [Valladares A, Muro-Pastor A M, Fillat M F, Herrero A, Flores E. “Constitutive and nitrogen-regulated promoters of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacterium Anabaena sp.” FEBS Lett (1999). 449(2-3):159-64], the RbcS2 promoter of Chlamydomonas [Leon R, Couso I, Fernandez E. “Metabolic engineering of ketocarotenoids biosynthesis in the unicellular microalgae Chlamydomonas reinhardtii.” J Biotechnol (2007). 130(2):143-152], and the core promoter sequence of psbA from Microcystis aeruginosa K-81 [Shibato J, Asayama M, Shirai M. “Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors.” Biochim. Biophys Acta (1998). 1442(2-3):296-303].

As necessary, after designing and testing pathways, the strength of constitutive promoters are “tuned” to increase or decrease levels of transcription to optimize a network, for example, by modifying the conserved −35 and −10 elements or the spacing between these elements [Alper H, Fischer C, Nevoigt E, Stephanopoulus G. “Tuning genetic control through promoter engineering.” PNAS (2005). 102(36):12678-12783; Jensen P R and Hammer K. “The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters.” Appl Environ Microbiol (1998). 64(I):82-87; Mijakovic I, Petranovic D, Jensen P R. Tunable promoters in system biology. Curr Opin Biotechnol (2005). 16:329-335; De Mey M, Maertens J, Lequeux G J, Soetaert W K, Vandamme E J. “Construction and model-based analysis of a promoter library from E. coli: an indispensable tool for metabolic engineering.” BMC Biotechnology (2007) 7:34].

When constitutive expression proves non-optimal (i.e., has deleterious effects, is out of sync with the network, etc.) inducible promoters are used. Inducible promoters are “off” (not transcribed) prior to addition of an inducing agent, frequently a small molecule or metabolite. Examples of suitable inducible promoter systems include the arabinose inducible P_(bad) [Khlebnikov A, Datsenko K A, Skaug T, Wanner B L, Keasling J D. “Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.” Microbiology (2001). 147 (Pt 12): 3241-7], the rhamnose inducible rhaP_(BAD) promoter [Haldimann A, Daniels L, Wanner B. J Bacteriol (1998). “Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon.” 180:1277-1286], the propionate inducible pPRO [Lee S K and Keasling J D. “A propionate-inducible expression system for enteric bacteria.” Appl Environ Microbiol (2005). 71(11):6856-62)], the IPTG-inducible lac promoter [Gronenborn. Mol Gen Genet (1976). “Overproduction of phage lambda repressor under control of the lac promoter of Escherichia coli.” 148:243-250], the synthetic tac promoter [De Boer H A, Comstock L J, Vasser M. “The tac promoter: a functional hybrid derived from the trp and lac promoters.” PNAS (1983). 80:21-25], the synthetic trc promoter [Brosius J, Erfle M, Storella J. “Spacing of the −10 and −35 regions in the tac promoter. Effect on its in vivo activity.” J Biol Chem (1985). 260:3539-3541], or the T7 RNA polymerase system [Studier F W and Moffatt B A. “Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.” J Mol Biol (1986]. 189:113-130, the tetracycline or anhydrotetracycline-inducible tetA promoter/operator system [Skerra A. “Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli” Gene (1994). 151:131-135], the nickel inducible Cpx1 and Cyc6 promoters of Chlamydomonas reinhardtii [Quinn J M, Kropat J, Merchant S. “Copper response element and Crr1-dependent nickel-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii.” Eukaryotic Cell (2003). 2(5):995-1002], the nitrite-inducible nirA promoter of Synechococcus [Qi Q, Hao M, Ng W, Slater S C, Baszis S R, Weiss J D, and Valentin H E. “Application of the Synechococcus nirA promoter to establish an inducible expression system for engineering the Synechocystis tocopherol pathway.” Appl. Environ. Microb (2005) 71(10):5678-5684], the sulfur-responsive arylsulfatase promoter of Volvox [Hallman A and Sumper M. “Reporter genes and highly regulated promoters as tools for transformation experiments in Volvox carteri.” Proc Natl Acad Sci (1994). 91(24):11562-11566]. These and other naturally-occurring or synthetically-derived inducible promoters are employed (see, e.g., U.S. Pat. No. 7,235,385; Methods for enhancing expression of recombinant proteins).

Alternate origins of replication are selected to provide additional layers of expression control. The number of copies per cell contributes to the “gene dosage effect.” For example, the high copy pMB1 or colE1 origins are used to generate 300-1000 copies of each plasmid per cell, which contributes to a high level of gene expression. In contrast, plasmids encoding low copy origins, such as pSC101 or p15A, are leveraged to restrict copy number to about 1-20 copies per cell. Techniques and sequences to further modulate plasmid copy number are known (see, e.g., U.S. Pat. No. 5,565,333, Plasmid replication origin increasing the copy number of plasmid containing said origin; U.S. Pat. No. 6,806,066, Expression vectors with modified ColE1 origin of replication for control of plasmid copy number). Certain organisms, including Synechococcus sp. PCC 7002, encode endogenous plasmids with varying copy numbers [Yano S, Kawata Y, Kojima H. “Salinity-dependent copy number changes of endogenous plasmids in Synechococcus sp. PCC 7002]; thus, gene dosage can be modified by targeting expression cassettes to distinct endogenous plasmids.

Expression levels are also optimized by modulation of translation efficiency. In E. coli, a Shine-Dalgarno (SD) sequence [Shine J and Dalgarno L. Nature (1975) “Determination of cistron specificity in bacterial ribosomes.” 254(5495):34-8] is a consensus sequence that directs the ribosome to the mRNA and facilitates translation initiation by aligning the ribosome with the start codon. Modulation of the SD sequence is used to increase or decrease translation efficiency as appropriate [de Boer H A, Comstock L J, Hui A, Wong E, Vasser M. Gene Amplif Anal (1983). “Portable Shine-Dalgarno regions; nucleotides between the Shine-Dalgarno sequence and the start codon effect the translation efficiency”. 3: 103-16; Mattanovich D, Weik R, Thim S, Kramer W, Bayer K, Katinger H. Ann NY Acad Sci (1996). “Optimization of recombinant gene expression in Escherichia coli.” 782:182-90.]. Of note, a high level of translation can be observed in certain contexts in the absence of an SD sequence [Mutsuda M and Sugiura M. “Translation initiation of cyanobacterial rbcS mRNAs requires the 38-kDa ribosomal protein S1 but not the Shine-Dalgarno sequence.” J Biol Chem (2006). 281(50):38314-38321; Xu J, Mironova R, Ivanov I G, Abouhaidar M G. J Basic Microbiol (1999). “A polylinker-derived sequence, PL, highly increased translation efficiency in Escherichia coli.” 39(1):51-60]. Secondary mRNA structure is engineered in or out of the genes of interest to modulate expression levels [Klinkert B, Elles I, Nickelsen J. “Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary structure blocking the AUG start codon.” Nucleic Acids Res (2006). 34(1):384-94; Cebe R and Geiser M. Protein Expr Purif (2006). “Rapid and easy thermodynamic optimization of 5′-end of mRNA dramatically increases the level of wild type protein expression in Escherichia coli.” 45(2):374-80; Zhang W, Xiao W, Wei H, Zhang J, Tian Z. Biochem Biophys Res Commun (2006). “mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli.” 349(1):69-78; Voges D, Watzele M, Nemetz C, Wizemann S, Buchberger B. Biochem Biophys Res Commun (2004). “Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system.” 318(2):601-14]. Codon usage is also manipulated to increase or decrease levels of translation [Deng T. FEBS Lett (1997). “Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization.” 409(2):269-72; Hale R Sand Thompson G. Protein Expr Purif (1998). “Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli.” 12(2):185-8].

In some embodiments, each gene of interest is expressed on a unique plasmid. In preferred embodiments, the desired biosynthetic pathways are encoded on multi-cistronic plasmid vectors. Useful expression vectors are designed internally and synthesized by external gene synthesis providers.

Optimizations

The below biosynthetic pathways and modules are first tested and optimized using episomal plasmids described above. Non-limiting optimizations include promoter swapping and tuning, ribosome binding site manipulation, alteration of gene order (e.g., gene ABC versus BAC, CBA, CAB, BCA), co-expression of molecular chaperones, random or targeted mutagenesis of gene sequences to increase or decrease activity, folding, or allosteric regulation, expression of gene sequences from alternate species, codon manipulation, addition or removal of intracellular targeting sequences such as signal sequences, and the like.

Each gene or module is optimized individually, or alternately, in parallel. Functional promoter and gene sequences are subsequently integrated into the photoautotrophic host's chromosome to enable stable propagation in the absence of selective pressure (i.e., inclusion of antibiotics) using standard techniques known to those skilled in the art.

Table 2 lists genes which are downregulated or knocked-out to enhance carbon fixation rates, thermotolerance, pH tolerance, flue gas tolerance, salt tolerance, light harvesting efficiencies, reducing power generation, and nutrient independence, together with information on associated pathways, Enzyme Commission (EC) Numbers, exemplary gene names, source organism, and GenBank accession numbers.

Disruption of Endogenous DNA Sequences

In certain instances, chromosomal DNA sequence native (i.e., “endogenous”) to the host organism are altered. Manipulations are made to non-coding regions, including promoters, ribosome binding sites, transcription terminators, and the like to increase or decrease expression of specific gene product(s). In alternate embodiments, the coding sequence of an endogenous gene is altered to affect stability, folding, activity, or localization of the intended protein. Alternately, specific genes can be entirely deleted or “knocked-out.” Techniques and methods for such manipulations are known to those skilled in the art [Nelson J A, and Lefebvre P A. “Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.” Mol Cell Bio (1995). 15(10):5762-5769; Hanson T E and Tabita F R. “A ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress.” Proc Natl Acad Sci (2001). 98(8):4397-4402; Sugita C, Mutsuda M, Sugiura M, Sugita M. “Targeted deletion of genes for eukaryotic RNA-binding proteins, Rbp1 and Rbp2, in the cyanobacterium Synechococcus sp. Strain PCC7942: Rbp1 is indispensable for cell growth at low temperatures.” FEMS Microbiol Letters (1999). 176(1): 155-161; Kirilovsky D, Roncel M, Boussac A, Wilson A, Zurita J L, Ducruet J, Bottin H, Sugiura M, Ortega J M, Rutherford A W. “Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus. Study of Redox mutants.” J Biol Chem (2004). 279(51):52869-80; Datsenko K A, Wanner B L. PNAS (2000). “One-step inactivation of chromosomal genes in E. coli K-12 using PCR Products.” 97: 6640-6645; Link A J et al. J Bacteriol (1997). “Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization.” 179:6228-6237; Baba T et al. Mol Syst Biol (2006). Construction of Escherichia coli K-12 in-frame, single gene knockout mutants: the Keio collection.” 2:2006.0008; Tischer B K, von Einem J, Kaufer B, Osterrieder N. Biotechniques (2006). “Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli.” 40(2):191-7; McKenzie G J, Craig N L. BMC Microbiol (2006). Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event.” 6:39].

In certain embodiments, post-transcriptional gene silencing (PTGS) is employed to reduce the expression level of an endogenous gene via expression of a heterologous RNA sequence, frequently antisense to the gene requiring disruption [Lechtreck K, Rostmann J, and Grunow A. “Analysis of Chlamydomonas SF-assemblin by GFP tagging and expression of antisense constructs.” J. Cell Sci (2002). 115:1511-1522; Smith N A, Singh S P, Wang M, Stotjesdijk P A, Green A G, and Waterhouse P M. “Total silencing by intron-spliced hairpin RNAs.” Nature (2000). 407:319-320; Furhmann M, Stahlberg A, Govorunova E, Rank S, and Hegeman P. “The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses.” J. Cell Sci (2001). 114:3857-3863; Rohr J, Sarkar N, Balenger S Jeong B R, Cerutti H. “Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas” Plant J (2004). 40(4):611-21].]

In other embodiments, expression of naturally encoded or exogenous small RNA or microRNA species is employed to downregulate endogenous gene expression [Molnar A, Schwach F, Studholme D J, Tgyenemann E C, and Baulcombe D C. “miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii.” Nature (2007). 447(7148):1126-9; Zhao T, Li G, Mi S, Li S, Hannon G J, Wang X J, Qi Y. “A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii.” Genes Dev (2007). 21(10):1190-203].

Selections and Assays

Selective pressure provides a valuable means for testing and optimizing the HyperPhotosynthetic organisms. The ability to survive under ever increasing temperatures provides evidence for successful implementation of the improved thermotolerance module. The ability to grow in media bubbled with flue gas (or an artificial gas formulation that approximates flue gas composition) confirms the successful implementation of the improved flue gas tolerance module. The ability to replicate more rapidly than the wild-type counterparts confirms the successful implementation of the improved CO₂ fixation module. The ability to survive and replicate in media lacking Vitamin B₁₂ as a media supplement confirms the successful implementation of the Vitamin B₁₂ module.

If desired, additional genetic variation can be introduced prior to selective pressure by treatment with mutagens, such as ultra-violet light, alkylators [e.g., ethyl methanesulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES), and nitrosoguanidine (NTG, NG, MMG)], DNA intercalators (e.g., ethidium bromide), nitrous acid, base analogs, bromouracil, transposons, and the like.

Alternately or in addition to selective pressure, pathway activity can be monitored following growth under permissive (i.e., non-selective) conditions by measuring specific product output via various metabolic labeling studies (including radioactivity), biochemical analyses (Michaelis-Menten), gas chromatography-mass spectrometry (GC/MS), mass spectrometry, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), capillary electrophoresis (CE), and high pressure liquid chromatography (HPLC).

Fermentation Methods

The production and isolation of products from HyperPhotosynthetic organisms can be enhanced by employing specific fermentation techniques. An essential element to maximizing production while reducing costs is increasing the percentage of the carbon source that is converted to such products. Carbon atoms, during normal cellular lifecycles, go to cellular functions including producing lipids, saccharides, proteins, and nucleic acids. Reducing the amount of carbon necessary for non-product related activities can increase the efficiency of output production. This is achieved by first growing microorganisms to a desired density. A preferred density would be that achieved at the peak of the log phase of growth. At such a point, replication checkpoint genes can be harnessed to stop the growth of cells. Specifically, quorum sensing mechanisms (reviewed in Camilli, A. and Bassler, B. L Science 311:1113; Venturi, V. FEMS Microbio Rev 30: 274; and Reading, N. C. and Sperandio, V. FEMS Microbiol Lett 254:1) can be used to activate genes such as p53, p21, or other checkpoint genes. Genes that can be activated to stop cell replication and growth in E. coli include umuDC genes, the overexpression of which stops the progression from exponential phase to stationary growth (Murli, S., Opperman, T., Smith, B. T., and Walker, G. C. 2000 Journal of Bacteriology 182: 1127.). UmuC is a DNA polymerase that can carry out translesion synthesis over non-coding lesions—the mechanistic basis of most UV and chemical mutagenesis. The umuDC gene products are required for the process of translesion synthesis and also serve as a DNA damage checkpoint. UmuDC gene products include UmuC, UmuD, umuD′, UmuD′₂C, UmuD′₂ and UmuD₂. Simultaneously, the product synthesis genes are activated, thus minimizing the need for critical replication and maintenance pathways to be used while the product is being made.

Alternatively, cell growth and product production can be achieved simultaneously. In this method, cells are grown in bioreactors with a continuous supply of inputs and continuous removal of product. Batch, fed-batch, and continuous fermentations are common and well known in the art and examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol (1992), 36:227.

In all production methods, inputs include carbon dioxide, water, and light. The carbon dioxide can be from the atmosphere or from concentrated sources including offgas or flue gas from coal plants, refineries, cement production facilities, natural gas facilities, breweries, and the like. Water can be no-salt, low-salt, marine, or high salt. Light can be solar or from artificial sources including incandescent lights, LEDs, fiber optics, and fluorescent lights.

Light-harvesting organisms are limited in their productivity to times when the solar irradiance is sufficient to activate their photosystems. In a preferred light-harvesting organism bioprocess, cells are enabled to grow and produce product with light as the energetic driver. When there is a lack of sufficient light, cells can be induced to minimize their central metabolic rate. To this end, the inducible promoters specific to product production can be heavily stimulated to drive the cell to process its energetic stores in the product of choice. With sufficient induction force, the cell will minimize its growth efforts, and use its reserves from light harvest specifically for product production. Nonetheless, net productivity is expected to be minimal during periods when sufficient light is lacking as no to few photons are net captured.

In a preferred embodiment, the cell is engineered such that the final product is released from the cell. In embodiments where the final product is released from the cell, a continuous process can be employed. In this approach, a reactor with organisms producing desirable products can be assembled in multiple ways. In one embodiment, the reactor is operated in bulk continuously, with a portion of media removed and held in a less agitated environment such that an aqueous product will self-separate out with the product removed and the remainder returned to the fermentation chamber. In embodiments where the product does not separate into an aqueous phase, media is removed and appropriate separation techniques (e.g., chromatography, distillation, etc.) are employed.

In an alternate embodiment, the product is not secreted by the cells. In this embodiment, a batch-fed fermentation approach is employed. In such cases, cells are grown under continued exposure to inputs (light, water, and carbon dioxide) as specified above until the reaction chamber is saturated with cells and product. A significant portion to the entirety of the culture is removed, the cells are lysed, and the products are isolated by appropriate separation techniques (e.g., chromatography, distillation, filtration, centrifugation, etc.).

In a preferred embodiment, the fermentation chamber will enclose a fermentation that is undergoing a continuous reductive fermentation. In this instance, a stable reductive environment is created. The electron balance is maintained by the release of carbon dioxide (in gaseous form). Augmenting the NAD/H and NADP/H balance, as described above, also can be helpful for stabilizing the electron balance.

Detection and Analysis of Gene and Cell Products

Any of the standard analytical methods, such as gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry, HPLC, capillary electrophoresis, Matrix-Assisted Laser Desorption Ionization time of flight-mass spectrometry, etc., can be used to analyze the levels and the identity of the product produced by the modified organisms of the present invention.

The ability to detect formation of a new, functional biochemical pathway in the HyperPhotosynthetic cell is important to the practice of the subject methods. In general, the assays are carried out to detect heterologous biochemical transformation reactions of the host cell that produce, for example, small organic molecules and the like as part of a de novo synthesis pathway, or by chemical modification of molecules ectopically provided in the host cell's environment. The generation of such molecules by the host cell can be detected in “test extracts,” which can be conditioned media, cell lysates, cell membranes, or semi-purified or purified fractionation products thereof. The latter can be, as described above, prepared by classical fractionation/purification techniques, including phase separation, chromatographic separation, or solvent fractionation (e.g., methanol ethanol, acetone, ethyl acetate, tetrahydrofuran (THF), acetonitrile, benzene, ether, bicarbonate salts, dichloromethane, chloroform, petroleum ether, hexane, cyclohexane, diethyl ether and the like). Where the assay is set up with a responder cell to test the effect of an activity produced by the host cell on a whole cell rather than a cell fragment, the host cell and test cell can be co-cultured together (optionally separated by a culture insert, e.g. Collaborative Biomedical Products, Bedford, Mass., Catalog #40446).

In certain embodiments, the assay is set up to directly detect, by chemical or photometric techniques, a molecular species which is produced (or destroyed) by a biosynthetic pathway of the recombinant host cell. Such a molecular species' production or degradation must be dependent, at least in part, on expression of the heterologous genomic DNA. In other embodiments, the detection step of the subject method involves characterization of fractionated media/cell lysates (the test extract), or application of the test extract to a biochemical or biological detection system. In other embodiments, the assay indirectly detects the formation of products of a heterologous pathway by observing a phenotypic change in the host cell, e.g. in an autocrine fashion, which is dependent on the establishment of a heterologous biosynthetic pathway in the host cell.

In certain embodiments, analogs related to a known class of compounds are sought, as for example analogs of alkaloids, aminoglycosides, ansamacrolides, beta-lactams (including penicillins and cephalosporins), carbapenems, terpinoids, prostanoid hormones, sugars, fatty acids, lincosaminides, macrolides, nitrofurans, nucleosides, oligosaccharides, oxazolidinones, peptides and polypeptides, phenazines, polyenes, polyethers, quinolones, tetracyclines, streptogramins, sulfonamides, steroids, vitamins and xanthines. In such embodiments, if there is an available assay for directly identifying and/or isolating the natural product, and it is expected that the analogs would behave similarly under those conditions, the detection step of the subject method can be as straightforward as directly detecting analogs of interest in the cell culture media or preparation of the cell. For instance, chromatographic or other biochemical separation of a test extract may be carried out, and the presence or absence of an analog detected, e.g., spectrophotometrically, in the fraction in which the known compounds would occur under similar conditions. In certain embodiments, such compounds can have a characteristic fluorescence or phosphorescence which can be detected without any need to fractionate the media and/or recombinant cell.

In related embodiments, whole or fractionated culture media or lysate from a recombinant host cell can be assayed by contacting the test sample with a heterologous cell (“test cell”) or components thereof. For instance, a test cell, which can be prokaryotic or eukaryotic, is contacted with conditioned media (whole or fractionated) from a recombinant host cell, and the ability of the conditioned media to induce a biological or biochemical response from the test cell is assessed. For instance, the assay can detect a phenotypic change in the test cell, as for example a change in: the transcriptional or translational rate or splicing pattern of a gene; the stability of a protein; the phosphorylation, prenylation, methylation, glycosylation or other post translational modification of a protein, nucleic acid or lipid; the production of 2nd messengers, such as cAMP, inositol phosphates and the like. Such effects can be measured directly, e.g., by isolating and studying a particular component of the cell, or indirectly such as by reporter gene expression, detection of phenotypic markers, and cytotoxic or cytostatic activity on the test cell.

When screening for bioactivity of test compounds produced by the recombinant host cells, intracellular second messenger generation can be measured directly. A variety of intracellular effectors have been identified. For instance, for screens intended to isolate compounds, or the genes which encode the compounds, as being inhibitors or potentiators of receptor- or ion channel-regulated events, the level of second messenger production can be detected from downstream signaling proteins, such as adenylyl cyclase, phosphodiesterases, phosphoinositidases, phosphoinositol kinases, and phospholipases, as can the intracellular levels of a variety of ions.

In still other embodiments, the detectable signal can be produced by use of enzymes or chromogenic/fluorescent probes whose activities are dependent on the concentration of a second messenger, e.g., such as calcium, hydrolysis products of inositol phosphate, cAMP, etc.

Many reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art. Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol. 216:362-368); β-lactamase or GST.

Transcriptional control elements for use in the reporter gene constructs, or for modifying the genomic locus of an indicator gene include, but are not limited to, promoters, enhancers, and repressor and activator binding sites. Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, of contact between the cell surface protein and the effector protein that modulates the activity of the cell surface protein. Examples of such genes include, but are not limited to, the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485), such as c-fos. Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein. The transcriptional control elements that are preferred for use in the gene constructs include transcriptional control elements from immediate early genes, elements derived from other genes that exhibit some or all of the characteristics of the immediate early genes, or synthetic elements that are constructed such that genes in operative linkage therewith exhibit such characteristics. The characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.

In still other embodiments, the detection step is provided in the form of a cell-free system, e.g., a cell-lysate or purified or semi-purified protein or nucleic acid preparation. The samples obtained from the recombinant host cells can be tested for such activities as inhibiting or potentiating such pairwise complexes (the “target complex”) as involving protein-protein interactions, protein-nucleic acid interactions, protein-ligand interactions, nucleic acid-nucleic acid interactions, and the like. The assay can detect the gain or loss of the target complexes, e.g. by endogenous or heterologous activities associated with one or both molecules of the complex.

Assays that are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target when contacted with a test sample. Moreover, the effects of cellular toxicity and/or bioavailability of the test sample can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the sample on the molecular target as may be manifest in an alteration of binding affinity with other molecules or changes in enzymatic properties (if applicable) of the molecular target. Detection and quantification of the pairwise complexes provides a means for determining the test samples efficacy at inhibiting (or potentiating) formation of complexes. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test sample. Moreover, a control assay can also be performed to provide a baseline for comparison. For instance, in the control assay conditioned media from untransformed host cells can be added.

The amount of target complex may be detected by a variety of techniques. For instance, modulation in the formation of complexes can be quantitated using, for example, detectably labeled proteins or the like (e.g., radiolabeled, fluorescently labeled, or enzymatically labeled), by immunoassay, or by chromatographic detection.

In still other embodiments, a purified or semi-purified enzyme can be used to assay the test samples. The ability of a test sample to inhibit or potentiate the activity of the enzyme can be conveniently detected by following the rate of conversion of a substrate for the enzyme.

In yet other embodiments, the detection step can be designed to detect a phenotypic change in the host cell which is induced by products of the expression of the heterologous genomic sequences. Many of the above-mentioned cell-based assay formats can also be used in the host cell, e.g., in an autocrine-like fashion.

In addition to providing a basis for isolating biologically-active molecules produced by the recombinant host cells, the detection step can also be used to identify genomic clones which include genes encoding biosynthetic pathways of interest. Moreover, by iterative and/or combinatorial sub-cloning methods relying on such detection steps, the individual genes which confer the detected pathway can be cloned from the larger genomic fragment.

The subject screening methods can be carried in a differential format, e.g. comparing the efficacy of a test sample in a detection assay derived with human components with those derived from, e.g., fungal or bacterial components. Thus, selectivity as a bacteriocide or fungicide can be a criterion in the selection protocol.

The host strain need not produce high levels of the novel compounds for the method to be successful. Expression of the genes may not be optimal, global regulatory factors may not be present, or metabolite pools may not support maximum production of the product. The ability to detect the metabolite will often not require maximal levels of production, particularly when the bioassay is sensitive to small amounts of natural products. Thus initial submaximal production of compounds need not be a limitation to the success of the subject method.

Finally, as indicated above, the test sample can be derived from, for example, conditioned media or cell lysates. With regard to the latter, it is anticipated that in certain instances there may be heterologously-expressed compounds that may not be properly exported from the host cell. There are a variety of techniques available in the art for lysing cells. A preferred approach is another aspect of the present invention, namely, the use of a host cell-specific lysis agent. For instance phage (e.g., P1, λ, φ80) can be used to selectively lyse E coli. Similarly, cyanophages can be used to selectively lyse cyanobacteria, such as Synechococcus and Prochlorococcus. Addition of such phage to grown cultures of host cells can maximize access to the heterologous products of new biosynthetic pathways in the cell. Moreover, such agents do not interfere with the growth of a tester organism, e.g., a human cell, that may be co-cultured with the host cell library.

Metabolic Optimization

As part of the optimization process, the invention also provides steps to eliminate undesirable side reactions, if any, that may consume carbon and energy but do not produce useful products (such as hydrocarbons, wax esters, surfactants and other hydrocarbon products). These steps may be helpful in that they can help to improve yields of the desired products.

A combination of different approaches may be used. Such approaches include, for example, metabolomics (which may be used to identify undesirable products and metabolic intermediates that accumulate inside the cell), metabolic modeling and isotopic labeling (for determining the flux through metabolic reactions contributing to hydrocarbon production), and conventional genetic techniques (for eliminating or substantially disabling unwanted metabolic reactions). For example, metabolic modeling provides a means to quantify fluxes through the cell's metabolic pathways and determine the effect of elimination of key metabolic steps. In addition, metabolomics and metabolic modeling enable better understanding of the effect of eliminating key metabolic steps on production of desired products.

To predict how a particular manipulation of metabolism affects cellular metabolism and synthesis of the desired product, a theoretical framework was developed to describe the molar fluxes through all of the known metabolic pathways of the cell. Several important aspects of this theoretical framework include: (i) a relatively complete database of known pathways, (ii) incorporation of the growth-rate dependence of cell composition and energy requirements, (iii) experimental measurements of the amino acid composition of proteins and the fatty acid composition of membranes at different growth rates and dilution rates and (iv) experimental measurements of side reactions which are known to occur as a result of metabolism manipulation. These new developments allow significantly more accurate prediction of fluxes in key metabolic pathways and regulation of enzyme activity. (Keasling, J. D. et al., “New tools for metabolic engineering of Escherichia coli,” In Metabolic Engineering, Publisher Marcel Dekker, New York, Nym 1999; Keasling, J. D, “Gene-expression tools for the metabolic engineering of bacteria,” Trends in Biotechnology, 17, 452-460, 1999; Martin, V. J. J., et al., “Redesigning cells for production of complex organic molecules,” ASM News 68, 336-343 2002; Henry, C. S., et al., “Genome-Scale Thermodynamic Analysis of Escherichia coli Metabolism,” Biophys. J., 90, 1453-1461, 2006.)

Such types of models have been applied, for example, to analyze metabolic fluxes in organisms responsible for enhanced biological phosphorus removal in wastewater treatment reactors and in filamentous fungi producing polyketides. See, for example, Pramanik, et al., “A stoichiometric model of Escherichia coli metabolism: incorporation of growth-rate dependent biomass composition and mechanistic energy requirements.” Biotechnol. Bioeng. 56, 398-421, 1997; Pramanik, et al., “Effect of carbon source and growth rate on biomass composition and metabolic flux predictions of a stoichiometric model.” Biotechnol. Bioeng. 60, 230-238, 1998; Pramanik et al., “A flux-based stoichiometric model of enhanced biological phosphorus removal metabolism.” Wat. Sci. Tech. 37, 609-613, 1998; Pramanik et al., “Development and validation of a flux-based stoichiometric model for enhanced biological phosphorus removal metabolism.” Water Res. 33, 462-476, 1998.

Products

The HyperPhotosynthetic organisms in the present invention may be engineered to yield product categories, including but not limited to, biological sugars, hydrocarbon products, solid forms, and pharmaceuticals.

Biological sugars include but are not limited to glucose, starch, cellulose, hemicellulose, glycogen, xylose, dextrose, fructose, lactose, fructose, galactose, uronic acid, maltose, and polyketides. In preferred embodiments, the biological sugar may be glycogen, starch, or cellulose.

Cellulose is the most abundant form of living terrestrial biomass (Crawford, R. L. 1981. Lignin biodegradation and transformation, John Wiley and Sons, New York.). Cellulose, especially cotton linters, is used in the manufacture of nitrocellulose. Cellulose is also the major constituent of paper. Cellulose monomers (beta-glucose) are linked together through 1,4 glycosidic bonds. Cellulose is a straight chain (no coiling occurs). In microfibrils, the multiple hydroxide groups hydrogen-bond with each other, holding the chains firmly together and contributing to their high tensile strength. Given a cellulose material, the portion that does not dissolve in a 17.5% solution of sodium hydroxide at 20° C. is Alpha cellulose, which is true cellulose; the portion that dissolves and then precipitates upon acidification is Beta cellulose, and the proportion that dissolves but does not precipitate is Gamma cellulose. Hemicellulose is a class of plant cell-wall polysaccharide that can be any of several heteropolymers. These include xylane, xyloglucan, arabinoxylan, arabinogalactan, glucuronoxylan, glucomannan, and galactomannan. This class of polysaccharides is found in almost all cell walls along with cellulose. Hemicellulose is lower in weight than cellulose, and cannot be extracted by hot water or chelating agents, but can be extracted by aqueous alkali. Polymeric chains bind pectin and cellulose, forming a network of cross-linked fibers.

There are essentially three types of hydrocarbon products: (1) aromatic hydrocarbon products, which have at least one aromatic ring; (2) saturated hydrocarbon products, which lack double, triple or aromatic bonds; and (3) unsaturated hydrocarbon products, which have one or more double or triple bonds between carbon atoms. A “hydrocarbon product” may be further defined as a chemical compound that consists of C, H, and optionally O, with a carbon backbone and atoms of hydrogen and oxygen, attached to it. Oxygen may be singly or double bonded to the backbone and may be bound by hydrogen. In the case of ethers and esters, oxygen may be incorporated into the backbone, and linked by two single bonds, to carbon chains. A single carbon atom may be attached to one or more oxygen atoms. Hydrocarbon products may also include the above compounds attached to biological agents including proteins, coenzyme A and acetyl coenzyme A. Hydrocarbon products include, but are not limited to, hydrocarbons, alcohols, aldehydes, carboxylic acids, ethers, esters, carotenoids, and ketones.

Hydrocarbon products also include alkanes, alkenes, alkynes, dienes, isoprenes, alcohols, aldehydes, carboxylic acids, surfactants, wax esters, polymeric chemicals [polyphthalate carbonate (PPC), polyester carbonate (PEC), polyethylene, polypropylene, polystyrene, polyhydroxyalkanoates (PHAs), poly-beta-hydroxybutryate (PHB), polylactide (PLA), and polycaprolactone (PCL)], monomeric chemicals [propylene glycol, ethylene glycol, and 1,3-propanediol, ethylene, acetic acid, butyric acid, 3-hydroxypropanoic acid (3-HPA), acrylic acid, and malonic acid], and combinations thereof. In some preferred embodiments, the hydrocarbon products are alkanes, alcohols, surfactants, wax esters and combinations thereof. Other hydrocarbon products include fatty acids, acetyl-CoA bound hydrocarbons, acetyl-CoA bound carbohydrates, and polyketide intermediates.

Recombinant organisms can be engineered to produce hydrocarbon products and intermediates over a large range of sizes. Specific alkanes that can be produced include, for example, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, and octadecane. In preferred embodiments, the hydrocarbon products are octane, decane, dodecane, tetradecane, and hexadecane. Hydrocarbon precursors such as alcohols that can be produced include, for example, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, and octadecanol. In more preferred embodiments, the alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, and decanol.

Surfactants are used in a variety of products, including detergents and cleaners, and are also used as auxiliaries for textiles, leather and paper, in chemical processes, in cosmetics and pharmaceuticals, in the food industry and in agriculture. In addition, they may be used to aid in the extraction and isolation of crude oils which are found hard to access environments or as water emulsions. There are four types of surfactants characterized by varying uses. Anionic surfactants have detergent-like activity and are generally used for cleaning applications. Cationic surfactants contain long chain hydrocarbons and are often used to treat proteins and synthetic polymers or are components of fabric softeners and hair conditioners. Amphoteric surfactants also contain long chain hydrocarbons and are typically used in shampoos. Non-ionic surfactants are generally used in cleaning products.

Hydrocarbons can additionally be produced as biofuels. A biofuel is any fuel that derives from a biological source—recently living organisms or their metabolic byproducts, such as manure from cows. A biofuel may be further defined as a fuel derived from a metabolic product of a living organism. Preferred biofuels include, but are not limited to, biodiesel, biocrude, ethanol, “renewable petroleum,” butanol, and propane.

Solid forms of carbon including, for example, coal, graphite, graphene, cement, carbon nanotubes, carbon black, diamonds, and pearls. Pure carbon solids such as coal and diamond are the preferred solid forms.

Pharmaceuticals can be produced including, for example, isoprenoid-based taxol and artemisinin, or oseltamivir.

EXAMPLES Plasmid Constructions

Construction of pJB5 Base Plasmid

The pJB5 base plasmid was designed as an empty expression vector for recombination into Synechococcus sp. PCC 7002. Two regions of homology, the Upstream Homology Region (UHR) and the Downstream Homology Region (DHR) were designed to flank the cloned gene(s) of interest. These 500 bp regions of homology correspond to positions 3301-3800 and 3801-4300 on the natural plasmid pAQ1 (Genbank Accession NC_(—)005025) for UHR and DHR respectively. The aadA promoter, gene sequence, and terminator were designed to confer spectinomycin and streptomycin resistance to the integrated construct. For expression, pJB5 was designed with the aph2 kanamycin resistance cassette promoter and ribosome binding site (RBS). Downstream of this promoter and RBS, we designed and inserted the restriction endonuclease recognition site for NdeI and EcoRI, as well as the sites for XhoI, BamHI, SpeI and PacI. Following the EcoRI site, the natural terminator from the alcohol dehydrogenase gene from Zymomonas mobilis (adhII) terminator was included. Convenient XbaI restriction sites flank the UHR and the DHR allowing cleavage of the DNA intended for recombination from the rest of the vector. pJB5, pJB6 and pJB7 were constructed by DNA2.0 (Menlo Park, Calif.).

Construction of pJB161 Base Plasmid

The pJB161 base plasmid was designed to complement the pJB5 base plasmid, but with different integration sites and resistance markers to allow for integration of two genes simultaneously into Synechococcus sp. PCC 7002. The UHR and the DHR from pJB5 were replaced with regions flanking the lactate dehydrogenase gene of Synechococcus sp. PCC 7002. The new ldh-UHR and ldh-DHR were amplified by PCR and correspond to positions 185990-77 and 184333-184913 on the natural plasmid pAQ7 (Genbank Accession NC_(—)0104774) for lac-UHR and lac-DHR respectively. The primers used for the PCR were as follows: Forward Primer for lac-UHR—ttgctacctgcagggccaccacagccaaattcatcgtt (SEQ ID NO: 1), Downstream Primer for lac-UHR—ggttgtgcggccgcagtattggctgtgatgttgg (SEQ ID NO: 2); Upstream Primer for lac-DHR—cgataaggcgcgccgaaactgcgccaagaatagc (SEQ ID NO: 3), Downstream Primer for lac-DHR—gtgtatggccggccatcgcctttatggtgctttatgtg (SEQ ID NO: 4). The Upstream Primer for lac-UHR added a SbfI restriction endonuclease site, the Downstream Primer for lac-UHR added a NotI restriction site, the Upstream Primer for lac-DHR added an AscI restriction site, and the Downstream Primer for lac-DHR added an FseI restriction site. The homology regions were amplified from Synechococcus sp. PCC 7002 genomic DNA using the high fidelity Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.). The amplified ldh-DHR region and the pJB5 plasmid were digested individually with FseI and AscI (New England Biolabs) restriction endonucleases using well known laboratory techniques. The resulting DNA fragments were gel isolated on a 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 (EpiCentre®) chemically competent cells using standard techniques, and confirmed by PCR. The resulting plasmid was subjected to another round of cloning to integrate the ldh-UHR region. The amplified ldh-UHR region and the newly constructed plasmid were digested individually with SbfI and NotI (New England Biolabs) restriction endonucleases using well known laboratory techniques. Both digestions were gel isolated on a 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 chemically competent cells using standard techniques (EpiCenter), and confirmed by PCR. The resulting plasmid, pJB165, was confirmed by PCR and restriction digestion.

Finally, to change the resistance marker for the integrated construct, a kanamycin resistance cassette, from the cloning vector pMAKK76 (Accession Number: U08460) was designed with restriction sites for PacI and AscI flanking the 5′ and 3′ end of the gene respectively. The cassette was constructed by DNA2.0 (Menlo Park, Calif.). This cassette, along with pJB165 were both individually digested with PacI and AscI (New England Biolabs) restriction endonucleases using well known laboratory techniques. Both digestions were gel isolated on a 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 chemically competent cells using standard techniques (EpiCentre). The resulting plasmid, pJB161, was confirmed by PCR and by resistance of transformed colonies to the antibiotic kanamycin.

Construction of pJB5-PdcAdhII and JCC136

The pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhII) genes were cloned into the pJB5 plasmid with the following procedure. The pdc-adhII genes from Zymomonas mobilis (Genbank: DD161475, M15394) were designed with an NdeI site replacing the start of the pdc coding region. Following the pdc gene, we designed two restriction endonuclease sites (XhoI and BamHI). Next, the adhII sequence was designed in whole subsequent to the restriction sites, and finally, the natural adhII terminator was included as well, downstream of an inserted EcoRI site. This construct was constructed by DNA2.0 (Menlo Park, Calif.) and was inserted by restriction digest with NdeI and EcoRI (New England Biolabs; Ipswitch, Mass.) on both pJB5 and the insert followed by ligation with a Quick Ligation Kit (New England Biolabs; Ipswitch, Mass.). The ligated construct was transformed into The NEB 5-alpha F′Iq Competent E. coli (High Efficiency) (New England Biolabs: Ipswitch, Mass.).

Transformation of pJB5-PdcAdhII into Synechococcus sp. PCC 7002 Using Standard Procedures.

Briefly, Synechococcus sp. PCC 7002 was grown for 48 h from colonies in an incubated shaker flask at 30° C. with 1% CO₂ to an OD₇₃₀ of 1.0 in A⁺ medium described in Frigaard N U et al. (2004) “Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation” Methods Mol Biol 274:325-340. Five hundred μL of culture was added to a test-tube with 30 μL of 1-5 μg of DNA prepped from a Qiagen Qiaprep Spin Miniprep Kit (Valencia, Calif.) for each construct. Cells were incubated bubbling in 1% CO₂ at approximately 1 bubble every 2 seconds for 4 hours. 200 μL of cells were plated on A⁺ medium plates with 1.5% Bacto-agar and grown at 30° C. for two days in low light. Spectinomycin was underlayed to give a final concentration of 10 μg/mL. Resistant colonies were visible in 7-10 days. Colonies were screened by PCR. The resulting strain was named JCC136.

Construction of pJB263 Base Plasmid

The pJB263 base plasmid was constructed to be similar to pJB5, but for integration and replacement of the glgA gene (Accession Number: NP_(—)441947) in Synechocystis sp. PCC 6803. The glgA-UHR and glgA-DHR were designed to 750 bp upstream and downstream of the glgA gene in Synechocystis sp. PCC 6803, which corresponds to (−) strands of 2266647 to 22667396 and 2264463 to 2265212 respectively on the chromosome (Accession Number: NC_(—)000911). The glgA-UHR and glgA-DHR were designed with flanking SbfI and NotI restriction endonuclease sites, as well as PacI and AscI restriction endonuclease sites, with a sequence spacer to ease later digest in between. This construct was constructed by DNA2.0 (Menlo Park, Calif.). This cassette, along with pJB5-PdcAdhII, was both individually digested with NotI and AscI (New England Biolabs) restriction endonucleases using well known laboratory techniques. Both digestions were gel isolated on a 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 chemically competent cells using standard techniques (EpiCentre). The resulting plasmid, pJB263, was confirmed by PCR and by resistance of transformed colonies to the antibiotic spectinomycin.

Examples

The examples provided herein illustrate the invention in more detail. These examples are provided to enable those skilled artisans to help understand and practice various aspects of the invention and therefore should not be construed as limiting. Various modifications and extensions of the invention in addition to those described herein will become apparent to those skilled artisans and therefore such modifications and extensions fall within the scope of invention.

Example 1 Improved Light Capture

Photosynthetic organisms have evolved elaborate methods to efficiently capture light, which is often times limiting in their natural habitats. Eukaryotic photoautotrophic organisms encode a superfamily of chlorophyll and carotenoid-binding proteins known as the light-harvesting complexes (LHCs), which capture and transfer light energy to the photosynthetic reaction centers [Green B R and Durnford D G. “The chlorophyll-carotenoid proteins of oxygenic photosynthesis.” Ann Rev Plant Physiol Plant Mol Biol (1996). 47:685-714]. Chlorophyll molecules are specifically arranged in so called “antenna” structures; the number of chlorophyll molecules per reaction center can vary considerably encompassing upwards of 350 chlorophyll a and chlorophyll b molecules per reaction center for photosystem II (PSII) and 300 chlorophyll a molecules for photosystem I (PSI). Antennas provide a means to increase light absorption spectra without having to build an entirely new protein-based reaction center and accompanying electron transport system. Large antenna provides survival advantages to organisms in the wild; however, under conditions of high light intensity, they absorb excess photons which must be wastefully dissipated as fluorescence or heat. Failure to safely dissipate a singlet-state excited chlorophyll molecule can result in formation of singlet oxygen, which is an extremely damaging reactive oxygen species [Muller P, Li X, Niyogi K K. “Non-photochemical quenching. A response to excess light energy.” Plant Physiology (2001). 125:1558-66].

Organisms naturally increase or decrease their chlorophyll antenna size as an adaptive response to changing light conditions [Falkowski P G and Owens T G. “Light-shade adaptation.” Plant Physiol (1980). 66:592-595; Ballottari M, Dall'Osto L, Morosinotto T, and Bassi R. “Contrasting behavior of higher plant photosystem I and II antenna systems during acclimation.” J Biol Chem (2007). 282(12):8947-58].

Recently, it has been demonstrated that the normally dynamic antenna size of microalgae can be permanently truncated genetically via downregulation of tla1 [Polle J, Kanakagiri S, and Melis A. “tla1, a DNA insertional transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size.” Planta (2003). 217:49-59; Tetali S D, Mitra M, and Melis A. “Development of the light-harvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii is regulated by the novel Tla1 gene.” Planta (2007). 225:813-829] or the entire family of LHC proteins [Mussgnug J H, Thomas-Hall S, Rupprecht J, Foo A, Klassen V, McDowall A, Schenk P M, Kruse O, and Hankamer B. “Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion.” (2007). 5(6):802-14]. Strains possessing smaller antenna exhibit reduced cell shading and higher productivities specifically under high light fluxes.

Chlorosomes, the light-harvesting antenna of green sulfur and green non-sulfur phototrophic bacteria, are specialized lipoprotein compartments typically comprising bacteriochlorophyll (BChl) c, BChl a, carotenoids, and quinones [Frigaard N U, Li H, Milks K J, and Bryant D A. “Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes. J Bacteriol (2004). 186(3):636-53]. In the wild, chlorosomes provide green bacteria significant survival advantages, as they enable growth under extremely low light conditions. The antenna structure can be eliminated by inactivating the BChl c synthase (bchK) [Friggard N U, Voigt G D, and Bryant D A. “Chlorobium tepidum mutant lacking bacteriochlorophyll c made by inactivation of the bchK gene, encoding bacteriochlorophyll c synthase.” J Bacteriol (2002). 184(12):3368-76]. Such mutants replicate about 7-fold slower than their wild-type counterparts under low-light conditions, but are only partially impaired (˜2.3-fold) under higher light intensities.

Phycobilisomes, the light harvesting antenna of cyanobacteria and red algae, are primarily comprised of the phycobiliproteins phycoerythrin, phycocyanin, and allophycocyanin [Grossman A R, Schaefer M R, Chiang G G, and Collier J L. “The phycobilisome, a light-harvesting complex responsive to environmental conditions.” Microbiol Rev (1993). 57(3):725-49] Like the light harvesting antenna of plants, algae, and green bacteria, the phycobilisomes are entirely dispensable in cyanobacteria [Ughy B and Ajlani G. “Phycobilisome rod mutants in Synechocystis sp. Strain PCC 6803.” Microbiology (2004). 150:4147-4156; Ajlani G and Vernotte C. Construction and characterization of phycobiliprotein-less mutant of Synechocystis sp. PCC 6803.” Plant Mol Biol (1998). 37:577-580; Anderson A K and Toole C M. “A model for early events in the assembly pathway of cyanobacterial phycobilisomes.” Mol. Microbiol (1998). 30(3):467-74].

While photoautotrophic organisms invariably utilize photosynthetic reaction centers to convert photonic energy into chemical energy (in the form of ATP, via proton motive force (PMF)-driven ATP synthase) and reducing power (NADPH), no known photoautotrophic organisms employ light-activated proton translocation systems as exemplified by archaeal rhodopsin-like proteins (bacteriorhodopsin) [Oesterhelt D and Stoeckenius W. “Rhodopsin-like protein from the purple membrane of Halobacterium halobium.” Nature New Biol (1971). 233(39):149-52]. Nevertheless, related sequences have definitively been shown to mediate light-driven energy generation (photoheterotrophy) in bacteria [Beja O, Aravind L, Koonin E V, Suzuki M, Hadd A, Nguyen L P, Jovanovich S B, Gates C M, Feldman R A, Spudich J L, Spudich E N, and DeLong E F. “Bacterial rhodopsin: evidence for a new type of phototrophy in the sea.” Science (2000). 289:1902-6; Beja O, Spudich E N, Spudich J L, Leclerc M, DeLong E F. “Proterhodopsin phototrophy in the ocean.” Nature (2001). 411:786-9; de la Torre J R, Christianson L M, Beja O, Suzuki M T, Karl D M, Heidelberg J, and DeLong E F. “Proteorhodopsin genes are distributed among divergent marine bacterial taxa.” Proc Natl Acad. Sci (2003). 100(22):12830-5].

The present invention teaches that exogenous expression of one or more forms of light-powered proton pumps in a photoautotroph increases organism efficiency by providing a parallel means to convert photonic energy into PMF, which can be used to power active transport of molecules across membranes or generate ATP through an endogenous or exogenous ATP synthase. It has been estimated that transport consumes between 15-25% of all energy during cell growth [Stouthamer A H. “A theoretical study on the amount of ATP required for synthesis of microbial cell material.” Antonie Van Leeuwenhoek (1973). 39(3):545-65; Carruthers A. “Mechanisms for the facilitated diffusion of substrates across cell membranes.” Biochemistry (1991). 30(16):3898-906]. Expression of light-powered proton pumps thus provides photoautotrophic organisms with up to a 15-25% gain in energetic efficiency, which is manifested by an improvement in doubling-time, CO₂-fixation rates, and/or carbon-based product formation.

The proteorhodopsin (PR) gene is preferentially expressed in organisms. An exemplary PR sequence is locus ABL60988 described in Martinerz A, Bradley A S, Walbauer J R, Summons R E, DeLong E F. PNAS (2007). “Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host.” 104(13):5590-5595.

In addition, or as an alternative, a bacteriorhodopsin gene is expressed [Oesterhelt D, Stoeckenius W. Nature (1971) “Rhodopsin-like protein from the purple membrane of Halobacterium halobium.” 233:149-152]. An exemplary bacteriorhodopsin sequence is the NP_(—)280292 locus described in Ng W V et al. PNAS (2000). “Genome sequence of Halobacterium species NRC-1.” 97(22):12176-22181. Bacteriorhodopsin has previously been functionally expressed in yeast mitochondria [Hoffmann A, Hildebrandt V, Heberle J, Buldt G. “Photoactive mitochondria: In vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.” Proc. Natl. Acad. Sci (1994). 91: 9637-71].

Similarly, deltarhodopsin is expressed in addition to or as an alternative [Ihara K et al. J Mol Biol (1999). “Evolution of the archael rhodopsins: evolution rate changes by gene duplication and functional differentiation.” 285:163-174; Kamo N, Hashiba T, Kikukawa T, Araiso T, Ihara K, Nara T. Biochem Biophys Res Commun (2006). “A light-driven proton pump from Haloterrigena turkmenica: functional expression in Escherichia coli membrane and coupling with a H⁺ co-transporter.” 342(2): 285-90). An exemplary deltarhodopsin sequence is the AB009620 locus of Haloterrigena sp. Arg-4 described in Ihara K et al. J Mol Biol (1999). “Evolution of the archael rhodopsins: evolution rate changes by gene duplication and functional differentiation.” 285:163-174.

Similarly, the Leptosphaeria maculans opsin protein is expressed as an addition to or as an alternative to other proton pumps. An exemplary eukaryotic light-activated proton pump is opsin, accession AAG01180 from Leptosphaeria maculans, described in Waschuk S A, Benzerra A G, Shi L, and Brown L S. PNAS (2005). “Leptosphaeria rhodopsin: Bacteriorhodopsin-like proton pump from a eukaryote.” 102(19):6879-83].

Finally a xanthorhodopsin proton pump with a carotenoid antenna is expressed in addition to or as an alternative to other proton pumps (Balashov S P, Imasheva E S, Boichenko V A, Anton J, Wang J M, Lanyi J K. Science (2005) “Xanthorhodopsin: A proton pump with a light harvesting cartenoid antenna.” 309(5743): 2061-2064). An exemplary xanthorhodopsin sequence is locus ABC44767 from Salinibacter ruber DSM 13855 described in Mongodin E F et al. PNAS (2005). “The genome of Salinibacter ruber: Convergence and gene exchange among hyperhalophilic bacteria and archaea.” 102(50):18147-18152.

The pumps are used alone or in combination, optimized to the specific cell. The pumps can be directed to be incorporated into one or more than one membrane locations, for example the cytoplasmic, outer, mitochondrial, and/or chloroplast membranes. Xanthorhodopsin and proteorhodopsin co-expression represents an optimal combination.

In addition to the expression of one or more proton pumps described above, a retinal biosynthesis pathway is expressed. When PR and the retinal biosynthetic operon are functionally expressed in E. coli, the pump is able to restore proton motive force to azide-treated E. coli populations [Walter J M, Greenfield D, Bustamante C, Liphardt J. PNAS (2007). “Light-powering Escherichia coli with proteorhodopsin.” 104(7):2408-2412]. A six gene retinal biosynthesis operon, Accession number EF100190 is known (Martinerz A, Bradley A S, Walbauer J R, Summons R E, DeLong E F. PNAS (2007). “Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host.” 104(13):5590-5595) and encodes amino acid sequences Isopentenyl-diphosphate delta-isomerase (Idi), locus ABL60982; 15,15′-beta-carotene dioxygenase (Blh), locus ABL60983; Lycopene cyclase (CrtY), locus ABL60984; Phytoene synthase (CrtB), EC 2.5.1.32, locus ABL60985; Phytoene dehydrogenase (CrtI), locus ABL60986; and Geranylgeranyl pyrophosphate synthetase (CrtE), locus ABL60987.

The above 6 enzymes enable biosynthesis of retinal, which is the essential chromophore common to all rhodopsin-related proton pumps. In certain embodiments, additional spectral absorption is provided by carotenoids, as exemplified by the xanthorhodopsin pump and the C-40 salinixanthin antenna. In these embodiments, a beta-carotene ketolase (CrtO) is expressed, such as the crtO gene of the SRU_(—)1502 locus in Salinibacter ruber, described in Mongodin E F et al (2005). Other crtO genes include those from Rhodococcus erythropolis (AY705709) and Deinococcus radiodurans R1 (NP_(—)293819).

In certain embodiments, an endogenous or exogenous ATP synthase (EC 3.6.3.14) is overexpressed to enable maximal conversion of PMF into ATP. An exemplary ATP synthase is the F₁-F₀ ATP synthase from Escherichia coli. The membrane-bound F₀ subunit is comprised of amino acid sequences set forth in F0 sector of membrane-bound ATP synthase, subunit a (AtpB), locus NP_(—)418194; F0 sector of membrane-bound ATP synthase, subunit c (AtpE), locus NP_(—)418193; and F0 sector of membrane-bound ATP synthase, subunit b (AtpF), locus NP_(—)418192. The catalytic F₁ subunit is comprised of amino acid sequences set forth in F1 sector of membrane-bound ATP synthase, alpha subunit (AtpA), locus NP_(—)418190; F1 sector of membrane-bound ATP synthase, epsilon subunit (AtpC), locus NP_(—)418187; F1 sector of membrane-bound ATP synthase, beta subunit (AtpD), locus NP_(—)418188; F1 sector of membrane-bound ATP synthase, gamma subunit (AtpG), locus NP_(—)418189; and F1 sector of membrane-bound ATP synthase, delta subunit, (AtpH) locus NP_(—)418191.

In preferred embodiments, light-powered proton pumps are expressed in the context of one or more cellular membranes within an organism previously adapted, evolved, or engineered to contain smaller light harvesting antenna than the wild-type organism prior to adaptation, evolution, or engineering. Such organisms are uniquely efficient at converting light energy into cellular energy, biomass, and products, particularly when propagated under high light fluxes.

Expression of SAR86 Gene Encoding Proteorhodopsin for the Light Capture Module

The SAR86 proteorhodopsin gene (Beja, et al. (Science (2000) vol. 289: 1902-1906; Genbank: AF279106) as used herein was obtained from a plasmid previously constructed and provided by Jessica Walters and Jan Liphardt (University of California, Berkeley). The Walters-Liphardt host plasmid is a pBR322 plasmid derivative with a beta-lactamase cassette bearing the SAR86 proteorhodopsin gene. The proteorhodopsin gene was amplified from the Walters-Liphardt plasmid using PCR primers with the forward primer 5′-TATACATCATATGGGTAAATTATTACTGATATTAGGTAGTGTTATTGC-3′ (SEQ ID NO: 5) and the reverse primer 5′-GCTACAATTGTTAAGCATTAGAAGATTCTTTAACAGCAACATTCC-3′ (SEQ ID NO: 6). PCR amplifications were performed with the high fidelity Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.). The forward primer adds an NdeI restriction recognition site, and the reverse primer adds a stop codon and an MfeI restriction recognition site.

The amplified proteorhodpsin PCR gene product was cloned into the pJB5 expression vector (“pJB5-PR”) by digesting the insert and vector individually with NdeI and MfeI (New England Biolabs) restriction endonucleases with well known laboratory techniques. Both digestions were gel isolated on 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 chemically competent cells using standard techniques (EpiCenter), and confirmed by PCR.

pJB5-PR plasmid stocks were purified using Qiagen miniprep kit for transformation into Synechococcus sp. PCC 7002. One to two micrograms of pJB5-PR plasmid was added to Synechococcus sp PCC 7002 cells grown to an optical density of 1 and incubated at 37 C for 4 hours using low intensity orbital shaking and low level light source. Cells were then plated onto A⁺ solid media plates and placed in a lighted incubator (100-250 uE/m2/s, 37 C) for 1 day. Twenty-five micrograms/mL spectinomycin was underplayed on the plates and incubated until colonies grew (˜5 days). Integration into target Synechococcus host cells is confirmed by PCR of whole cell genomic DNA by a “colony PCR” protocol. Briefly, ˜1 mm colonies were resuspended in 50 μl deionized water, and 5 μl were used in 20 μl standard PCR reactions using Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.) with the addition of a 2 minute 98 C degree denaturation step at the very start of the standard PCR cycling conditions. The PCR showed correct bands for colonies, and the strain was named JCC1-SAR86 (FIG. 10, lane 5).

Example 2 Improving CO₂ Fixation

There are four known pathways that enable autotrophic carbon fixation: the 3-hydroxyproprionate (3-HPA) cycle (employed by Chloroflexus aurantiacus and Crenarchaeota symbiosum), the reductive TCA cycle (employed by Chlorobium tepidum), the reductive acetyl coenzyme A pathway (also known as Woods-Ljungdahl pathway; employed by chemolithoautotrophs such as Clostridium thermoaceticum, Methanobacterium thermautotrophicum, and Dusulfobacterium autotrophicum), and the reductive pentose phosphate cycle (also known as the Calvin cycle, employed by plants, algae, and all cyanobacteria). By definition, all photoautotrophic organisms possess the ability to fix inorganic CO₂ into complex, reduced organic carbon molecules, such as sugars.

The instant invention improves the rate and efficiency of CO₂ fixation by engineering functional improvements into the host's endogenous CO₂ fixation pathways, including overexpression of wild-type and/or variant enzymes. Alternately or in addition, functional improvements are engineered via supplementing the host's endogenous CO₂ fixation pathways with one or more exogenous CO₂ fixation enzymes or pathways.

The engineered organisms replicate more rapidly than their wild-type counterparts. Overexpression of two enzymes of the Calvin cycle in tobacco leaves has previously been shown to enhance growth compared to the wild-type plants [Tamoi M, Nagaoka M, Yabuta Y, and Shigeoka S. “Carbon metabolism in the Calvin cycle.” Plant Biotechnology (2005). 22:355-360]. While not wishing to be bound by theory, improved endogenous and/or exogenous CO₂ fixation enzymes enables more rapid conversion of CO₂ into biological intermediates and carbon products, which appears to be a limiting facet governing the doubling time of photoautotrophic organisms.

Table 1 lists genes which are overexpressed to enhance carbon fixation rates and efficiencies, together with information on associated pathways, Enzyme Commission (EC) Numbers, exemplary gene names, source organism, GenBank accession numbers, and homologs from alternate sources. When the parental organism encodes a gene with the indicated enzymatic activity, it is nevertheless useful to overexpress these components to improve CO₂ fixation. In one embodiment, the native enzyme sequence is overexpressed. In preferred embodiments, it is useful to overexpress an exogenous gene, which allows for more explicit regulatory control in the bioprocess and a means to potentially mitigate the effects of central metabolism regulation, which is focused around the native genes explicitly.

I. Enzymes for a Functional 3-Hydroxypropionate Cycle

The following enzyme activities are expressed to establish a functional 3-hydroxypropionate cycle. This pathway is natively employed by Chloroflexus aurantiacus [Herter S, Farfsing J, Gad'On N, Rieder C, Eisenreich W, Bacher A, and Fuchs G. J Bacteriol (2001). “Autotrophic CO₂ fixation by Chloroflexus aurantiacus: study of glyoxylate formation and assimilation via the 3-hydroxypropionate cycle.” 183(14):4305-16].

Acetyl-CoA carboxylase (ACCase), (EC 6.4.1.2), generates malonyl-CoA, ADP, and Pi from Acetyl-CoA, CO₂, and ATP. An exemplary ACCase subunit alpha is accA from E. coli, locus AAA70370. An exemplary ACCase subunit beta is accD from E. coli, locus AAA23807. An exemplary biotin-carboxyl carrier protein is accB from E. coli, locus ECOACOAC. An exemplary biotin carboxylase is accC from E. coli, locus AAA23748.

Malonyl-CoA reductase (also known as 3-hydroxypropionate dehydrogenase) (EC 1.1.1.59), generates 3-hydroxyproprionate, 2 NADP⁺, and CoA from malonyl-CoA and 2 NADPH. An exemplary bifunctional enzyme with both alcohol and dehydrogenase activities is mcr from Chloroflexus aurantiacus, locus AY530019.

3-hydroxypriopionyl-CoA synthetase (also known as 3-hydroxypropionyl-CoA dehydratase, or acryloyl-CoA reductase) generates propionyl-CoA, AMP, PPi (inorganic pyrophosphate), H₂O, and NADP⁺ from 3-hydroxypriopionate, ATP, CoA, and NADPH. An exemplary gene is propionyl-CoA synthase (pcs) from Chloroflexus aurantiacus, locus AF445079.

Propionyl-CoA carboxylase (EC 6.4.1.3) generates S-methylmalonyl-CoA, ADP, and Pi (inorganic phosphate) from Propionyl-CoA, ATP, and CO₂. An exemplary two subunit enzyme is propionyl-CoA carboxylase alpha subunit (pccA) from Roseobacter denitrificans, locus RD1_(—)2032 and propionyl-CoA carboxylase beta subunit (pccB) from Roseobacter denitrificans, locus RD1_(—)2028.

Methylmalonyl-CoA epimerase (EC 5.1.99.1) generates R-methylmalonyl-CoA from S-methylmalonyl-CoA. An exemplary enzyme from Rhodobacter sphaeroides is locus CP000661.

Methylmalonyl-CoA mutase (EC 5.1.99.2) generates succinyl-CoA from R-methylmalonyl-CoA. The yliK protein (locus NC000913.2) is an example.

Succinyl-CoA:L-malate CoA transferase generates L-malyl-CoA and succinate from succinyl-CoA and malate. An exemplary two subunit enzyme is SmtA from Chloroflexus aurantiacus, locus DQ472736.1 and SmtB from Chloroflexus aurantiacus, locus DQ472737.1.

Fumarate reductase (EC 1.3.1.6) generates fumarate and NADH from succinate and NAD⁺. An exemplary fumarate reductase is the E. coli frd operon (locus J01611). The frdA fumarate reductase flavoprotein subunit is known. It is important to note that some species may favor one direction over the other. Moreover, many of these proteins are present in organisms that express unidirectional and bidirectional versions. Examples are the frdB, fumarate reductase iron-sulfur subunit, and the g15 subunit the g13 subunit.

Fumarate hydratase (EC 4.2.1.2) generates malate from fumarate and water. E. coli encodes three distinct exemplary fumarate hydratases: the class I aerobic fumarate hydratase (fumA), locus CAA25204; the class I anaerobic fumarate hydratase (fumB), locus AAA23827; the class II fumarate hydratase (fumC), locus CAA27698.

L-malyl-CoA lyase (EC 4.2.1.2) generates acetyl-CoA and glyoxylate from L-malyl-CoA. An exemplary gene is mclA from Roseobacter denitrificans, locus NC_(—)008209.1.

The above enzyme activities, listed in this section, confer the ability to synthesize an organic 2-carbon glyoxylate molecule from 2 molecules of CO₂. The stoichiometry of this reaction is 2 CO₂+3 ATP+3 NADPH→Glyoxylate+2 ADP+2 Pi+AMP+PPi+3 NADP⁺.

II. Enzymes for a Functional Reductive TCA Cycle

The following enzyme activities are expressed to establish a functional reductive TCA cycle. This pathway is natively employed by Chlorobium tepidum.

ATP-citrate lyase (EC. 2.3.3.8) generates acetyl-CoA, oxaloacetate, ADP, and Pi from citrate, ATP, and CoA. An exemplary ATP citrate lyase is the two subunit enzyme from Chlorobium tepidum, comprising ATP citrate lyase subunit 1, locus CY1089 and ATP citrate lyase subunit 2, locus CT1088.

Hydrogenobacter thermophilus employs an alternate pathway to generate oxaloacetate from citrate. In a first step, the 2 subunit citryl-CoA synthetase generates citryl-CoA from citrate, ATP, and CoA. The large subunit: ccsA, locus BAD17844; the small subunit: ccsB, locus BAD17846.

The Hydrogenobacter thermophilus citryl-CoA ligase (ccl), locus BAD17841, generates oxaloacetate and acetyl-CoA from citryl-CoA.

Malate dehydrogenase (EC 1.1.1.37) generates malate and NAD⁺ from oxaloacetate and NADH. An exemplary malate dehydrogenase from Chlorobium tepidum is locus CAA56810.

Fumarase (also known as fumarate hydratase) (EC 4.2.1.2) generates fumarate and water from malate. E. coli encodes 3 different fumarase genes, which can be overexpressed in photoautotrophic organisms. An exemplary E. coli fumarase hydratase class I, (aerobic isozyme) is fumA. An exemplary E. coli fumarate hydratase class I (anaerobic isozyme) is fumB. An exemplary E. coli fumarate hydratase class II is fumC.

Succinate dehydrogenase (EC 1.3.99.1) generates succinate and FAD⁺ from fumarate and FADH₂ . E. coli encodes a four-subunit succinate dehydrogenase complex (SdhCDAB). These enzymes are also used in the 3-HPA pathway above, but in the reverse direction. It is important to note that some species may favor one direction or the other. Succinate dehydrogenase and fumarate reductase are reverse directions of the same enzymatic interconversion, succinate+FAD⁺→fumarate+FADH₂. In Escherichia coli, the forward and reverse reactions are catalyzed by distinct complexes: fumarate reductase operates under anaerobic conditions and succinate dehydrogenase operates under aerobic conditions. This group also includes a region of the B subunit of a cytosolic archaeal fumarate reductase, for example the SdhA flavoprotein subunit, locus NP_(—)415251; the SdhB iron-sulfur subunit, locus NP_(—)415252; the SdhC membrane anchor subunit, locus NP_(—)415249; and the SdhD membrane anchor subunit, locus NP_(—)415250.

Acetyl-CoA:succinate CoA transferase (also known as succinyl-CoA synthetase) (EC 6.2.1.5) generates succinyl-CoA, ADP, and Pi from succinate, CoA, and ATP. E. coli encodes a heterotetramer of two alpha and beta subunits. An exemplary E. coli succinyl-CoA synthetase subunit alpha is sucD, locus AAA23900. An exemplary E. coli succinyl-CoA synthetase subunit beta is sucC, locus AAA23899. Chlorobium tepidum sucC (AAM71626) and sucD (AAM71515) may also be used.

2-oxoketoglutarate synthase (also known as alpha-ketoglutarate synthase) (EC 1.2.7.3) generates alpha-ketoglutarate, CO₂, and oxidized ferredoxin from succinyl-CoA, CO₂, and reduced ferredoxin. An exemplary enzyme from Chlorobium limicola DSM 245 is a 4 subunit enzyme with accession numbers EAM42575; EAM42574; EAM42853; and EAM42852. This activity was functionally expressed in E. coli. Yun N R, Arai H, Ishii M, Igarashi Y. Biochem Biophys Res Communic (2001). The Genes for anabolic 2-oxoglutarate: Ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK6. 282 (2): 589-594. There is another 5-subunit OGOR cluster in the same bacterium. Yun N R et al. Biochem Biophys Res Communic (2002). A novel five-subunit-type 2-oxoglutalate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6. 292(1):280-6. The corresponding genes are forDABGE. An exemplary alpha-ketoglutarate synthase from Hydrogenobacter thermophilus is the heterodimeric enzyme that includes korA, locus AB046568:46-1869 and the korB locus AB046568:1883-2770.

Isocitrate dehydrogenase (EC 1.1.1.42) generates D-isocitrate and NADP+ from alpha-ketoglutarate, CO₂, and NADPH. An exemplary gene is the monomeric type idh from Chlorobium limicola, locus EAM42635. Another exemplary enzyme is that from Synechococcus sp WH 8102, icd, accession CAE06681.

In another embodiment, the NAD-dependent isocitrate dehydrogenase (EC 1.1.1.41) is expressed which generates isocitrate and NAD⁺ from alpha-ketoglutarate, CO₂, and NADH. An exemplary NAD-dependent enzyme is the two-subunit mitochondrial version from Saccharomyces cerevisiae. Subunit 1, idh1 locus YNL037C. The second subunit is idh2, locus YOR136W.

Aconitase (also known as aconitate hydratase or citrate hydrolyase) (EC 4.2.1.3) generates citrate from D-citrate via a cis-aconitate intermediate. E. coli encodes aconitate hydratase 1 and 2 (acnA and acnB). An exemplary aconitate hydrase 1 is E. coli acnA, locus b1276. An exemplary E. coli aconitate hydratase 2 is acnB, locus b0118.

Pyruvate synthase (also known as pyruvate:ferredoxin oxidoreductase) (EC 1.2.7.1) generates pyruvate, CoA, and an oxidized ferrodoxin from acetyl-CoA, CO₂, and a reduced ferredoxin. An exemplary pyruvate synthase is the tetrameric enzyme porABCD from Clostridium tetani E88, whereby subunit porA, locus AA036986; subunit porB, locus AA036985; subunit porC, locus AA036988; and subunit porD, locus AA036987.

Phosphoenolpyruvate synthase (also known as PEP synthase, pyruvate, water dikinase) (EC 2.7.9.2) generates phosphoenolpyruvate, AMP, and Pi from pyruvate, ATP, and water. E. coli encodes an exemplary PEP synthase, ppsA. The E. coli ppsA enzyme, locus AAA24319 and the corresponding enzyme from Aquifex aeolicus VF5 ppsA, locus AAC07865, may also be used.

Phosphoenolpyruvate carboxylase (also known as PEP carboxylase PEPCase, PEPC) (EC 4.1.1.31) generates oxaloacetate and Pi from phosphoenolpyruvate, water, and CO₂ . E. coli encodes an exemplary PEP carboxylase, ppC. The E. coli ppC enzyme, locus CAA29332 may also be used.

The above enzymes, described in this section, confer the ability to synthesize an organic 2-carbon acetyl-CoA molecule from 2 molecules of CO₂. The stoichiometry of this reaction is 2 CO₂+2 ATP+3 NADH+1 FADH₂+CoASH→acetyl-CoA+2 ADP+2 Pi+AMP+PPi+FAD⁺+3 NAD⁺.

III. Enzymes for a Functional Woods-Ljungdahl Cycle

The following enzyme activities are expressed in to establish a functional Woods-Ljungdahl pathway. This pathway is natively employed by Moorella thermoacetica (previously known as Clostridium thermoaceticum), Methanobacterium thermoautrophicum, and Desulfobacterium autotrophicum.

NADP-dependent formate dehydrogenase (EC 1.2.1.4.3) generates formate and NADP⁺ from CO₂ and NADPH. An exemplary NADP-dependent formate dehydrogenase is the two-subunit Mt-fdhA/B enzyme from Moorella thermoacetica (previously known as Clostridium thermoaceticum) which contains Mt-fdhA, locus AAB18330 and the beta subunit, Mt-fdhB, locus AAB18329.

Formate tetrahydrofolate ligase (EC 6.3.4.3) generates 10-formyltetrahydrofolate, ADP, and Pi from formate, ATP, and tetrahydrofolate. An exemplary formate tetrahydrofolate ligase is from Clostridium acidi-urici, locus M21507. Alternate sources for this enzyme activity include locus AAB49329 from Streptococcus mutans (Swiss-Prot entry Q59925), or the protein with Swiss-Prot entry Q8XHL4 from Clostridium perfringens encoded by the locus BA000016.

Methenyltetrahydrofolate cyclohydrolase (also known as 5,10-methylenetetrahydrofolate dehydrogenase) (EC 3.5.4.9 and 1.5.1.5) generates 5,10-methylene-THF, water, and NADP⁺ from 10-formyltetrahydrofolate and NADPH via a 5,10-methyenyltetrahydrofolate intermediate. E. coli encodes a bifunctional methenyltetrahydrofolate cyclohydrolase/dehydrogenase, folD for example, the E. coli enzyme, locus AAA23803. Alternate sources for this enzyme activity include locus ABC19825 (folD) from Moorella thermoacetica, locus AA036126 from Clostridium tetani; and locus BAB81529 from Clostridium perfringens. All are bifunctional folD enzymes.

Methylene tetrahydrofolate reductase (EC 1.5.1.20) generates 5-methyltetrahydrofolate and NADP⁺ from 5,10-methylene-trahydrofolate and NADPH. E. coli encodes an exemplary methylene tetrahydrofolate reductase, metF for example, the E. coli enzyme, locus CAA24747. Alternative sources for this enzyme activity include locus AAC23094 from Haemophilus influenzae and locus CAA30531 from Salmonella typhimurium.

5-methyltetrahydrofolate corrinoid/iron sulfur protein methyltransferase generates tetrahydrofolate and a methylated corrinoid Fe—S protein from 5-methyltetrahydrofolate and a corrinoid Fe—S protein. An exemplary gene, acsE, is encoded by locus AAA53548 in Moorella thermoacetica. This activity has been functionally expressed in E. coli (Roberts D L, Zhao S, Doukov T, and Ragsdale S. The reductive acetyl-CoA Pathway: Sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/Urib-sulfur protein methyltransferase from Clostridium thermoaceticum. J. Bacteriol (1994). 176(19):6127-30). Another source for this activity is encoded by the acsE gene from Carboxydothermus hydrogenoformas locus CP000141.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (EC 1.2.7.4/1.2.99.2 and 2.3.1.169) is a bifunctional two-subunit enzyme which generates acetyl-CoA, water, oxidized ferredoxin, and a corrinoid protein from CO₂, reduced ferredoxin, and a methylated corrinoid protein. An exemplary carbon monoxide dehydrogenase enzyme, subunit beta, is encoded by locus AAA23228 from Moorella thermoacetica. Another exemplary source of this activity is encoded by the acsB gene, locus CHY_(—)1222 from Carboxydothermus hydrogenoformase with protein accession YP_(—)360060. An exemplary acetyl-CoA synthase, subunit alpha, is locus AAA23229 from Moorella thermoacetica.

The above enzymes, described in this section, confer the ability to synthesize an organic 2-carbon acetyl-CoA molecule from 2 molecules of CO₂. The stoichiometry of this reaction is 2 CO₂+1 ATP+2 NADPH+2 reduced ferredoxins+coenzyme A→acetyl-CoA+2 H₂O+ADP+Pi+2 NADP⁺+2 oxidized ferredoxins.

Cells engineered to contain a functional CO₂ fixation pathway are selected for via growth in minimal media lacking an organic carbon source. In addition to survival-based selections, cells can be grown in minimal media in the presence of radiolabeled CO₂ (i.e., C¹⁴—CO₂). Detailed incorporation studies are employed to verify and characterize metabolic assimilation using common techniques known to those skilled in the art.

IV. Enzymes for a Functional Reductive Pentose Phosphate Cycle

The following enzyme activities are expressed to establish a functional reductive pentose phosphate (Calvin) cycle. This pathway is natively employed by all plants, algae, and cyanobacteria.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (EC 4.1.1.39) which productively generates two molecules of 3-phosphoglycerate from ribulose-1,5-bisphosphate, CO₂, and H2_(O). As its name suggests, RuBisCO can also catalyze a non-productive oxygenation reaction. Several classes of RuBisCO are known, any and all of which can be over-expressed [Watson G M F, Tabita F R. FEMS Microbiology Letters (1997) “Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation.” 146(1):13-22]. An exemplary Type I “Green-like” RuBisCO arising from the purple bacterial group comes from Synechococcus sp WH7803 (Ribulose-1,5-bisphosphate carboxylase/oxygenase—small subunit (CbbS), locus AAB48081) and (Ribulose-1,5-bisphosphate carboxylase/oxygenase—large subunit (CbbL), locus AAB8080). An exemplary Form I “Green-like” RuBisCO arising from the cyanobacterial/plant group comes from Synechococcus elongatus PCC 6301 (Ribulose-1,5-bisphosphate carboxylase/oxygenase—small subunit (RbcS), locus YP_(—)170839) and (Ribulose-1,5-bisphosphate carboxylase/oxygenase—large subunit (RbcL), locus YP_(—)170840). An exemplary Form I “Red-like” RuBisCO comes from Rhodobacter sphaeroides (Ribulose-1,5-bisphosphate carboxylase/oxygenase—small subunit (CbbS), locus P27998) and (Ribulose-1,5-bisphosphate carboxylase/oxygenase—large subunit (CbbL), locus P27997). An exemplary Form II RuBisCO comes from Rhodobacter sphaeroides as well (Ribulose-1,5-bisphosphate carboxylase/oxygenase (CbbM), locus P29278). Finally, an exemplary Form III RuBisCO comes from Methanocaldococcus jannaschii (Ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcL), locus Q58632).

In some embodiments, rubisco activase is overexpressed to improve the activation of RuBisCO and/or to release competitive inhibitors of RuBisCO carbon fixation. An exemplary rubisco activase is comes from Synechococcus sp. JA-3-3Ab (locus ABC98646).

Phosphoglycerate kinase (PGK) (EC 2.7.2.3) generates 1,3-bisphosphoglycerate ADP from 3-phosphoglycerate and ATP. An exemplary phosphoglycerate kinase is locus BAD78623 from Synechococcus sp. PCC 6301.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) generates glyceraldehyde 3-phosphate and NADP⁺ from 1,3-bisphosphoglycerate and NADPH. An exemplary GAPDH is encoded by the cbbG gene, locus NP_(—)875968 from Prochlorococcus marinus.

Triosephosphate isomerase (EC 5.3.1.1) generates dihydroxyacetone phosphate (DHAP) from glyceraldehyde 3-phosphate. An exemplary triosephosphate isomerase is encoded by the tpiA gene, locus Q59994 from Synechocystis sp. PCC 6803.

Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) generates fructose-1,6-bisphosphate from DHAP and glyceraldehyde 3-phosphate. An exemplary class I fructose 1,6-bisphosphate aldolase is encoded by the fda gene, locus NP_(—)441723 from Synechocystis sp. PCC 6803. An exemplary class II fructose 1,6-bisphosphate aldolase is encoded by the fbaA gene, locus BAA10184 from Synechocystis sp. PCC 6803.

Fructose-1,6-bisphosphatase (EC 3.1.3.11) generates fructose-6-phosphate and P, from fructose-1,6-bisphosphate and H₂0. An exemplary fructose-1,6-bisphosphatase is encoded by the fbp gene, locus NP_(—)441738 from Synechocystis sp. PCC 6803.

Transketolase (EC 2.2.1.1) generates xylulose 5-phosphate and erythrose 4-phosphate from fructose 6-phosphate and glyceraldehyde 3-phosphate. An exemplary transketolase is encoded by the tktA gene, locus YP_(—)171693 from Synechococcus sp. PCC 6301.

Pentose-5-phosphate-3-epimerase (EC 5.1.3.1) generates ribose-5-phosphate from xylulose-5-phosphate. An exemplary Pentose-5-phosphate-3-epimerase is encoded by the rpe gene, locus YP_(—)171630 from Synechocystis sp. PCC 6301.

Sedoheptulose-1,7-bisphosphate aldolase (EC 4.1.2.13) generates sedoheptulose-1,7-bisphosphate from erythrose-4-phosphate and DHAP. An exemplary Sedoheptulose-1,7-bisphosphate aldolase is encoded by the rpaA gene, locus NP_(—)681166 from Thermosynechococcus elongatus BP-1.

Sedoheptulose-1,7-bisphosphatase (SBPase) (EC 3.1.3.37) generates sedoheptulose-7-phosphate and Pi from sedoheptulose-1,7-bisphosphate and H₂O. An exemplary SBPase is encoded by the csbp gene, locus CAA52439 from Chlamydomonas reinhardtii.

Transketolase (EC 2.2.1.1) generates ribose 5-phosphate and xylulose 5-phosphate from sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate. An exemplary transketolase is encoded by the tktA gene, locus YP_(—)171693 from Synechococcus sp. PCC 6301.

Ribose 5-phosphate isomerase (EC 5.3.1.6) generates ribulose-5-phosphate from xylulose-5-phosphate or ribose-5-phosphate. An exemplary ribose-5-phosphate isomerase is encoded by the rpiA gene, locus YP_(—)171649 from Synechococcus elongatus PCC 6301.

Phosphoribulokinase (EC 2.7.1.19) generates ribulose-1,5-bisphosphate and ADP from ribulose-5-phosphate and ATP. An exemplary phosphoribulokinase is the prkA gene, locus AAA33090 from Chlamydomonas reinhardtii. In certain embodiments, the small CP12 protein is overexpressed, which regulates the Calvin cycle in via association/dissociation of PRK/CP12/GAPDH complexes depending on the ratio of NADPH/NADH [Tamoi M, Miyazaki T, Fukamizo T, Shigeoka S. “The calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions.” The Plant Journal (2005). 42:504-12]. An exemplary CP12 gene is locus BAC09372 from Thermosynechococcus elongatus BP-1.

In certain embodiments, carbon fixation rates and/or carbon product efficiencies are enhanced by altering carbon flux to specific key intermediates. In one embodiment, carbon flux is directed towards acetyl-CoA via overexpression of pantothenate kinase, such as locus YP_(—)473820 from Syenchococcus sp JA-3-3Ab and/or pyruvate dehydrogenase, such as pdhAB, locus YP_(—)1728660 (pdhA) and YP_(—)172072 (pdhB) from Synechococcus PCC 6301. In addition or as an alternative, endogenous genes that effectively reduce carbon flux through acetyl-CoA are downregulated or knocked-out. In these embodiments, acyl coenzyme A dehydrogenase (EC 1.3.99.3, locus YP_(—)171045), glycerol-3-phosphate dehydrogenase (EC 1.1.1.94, locus YP_(—)401539), and/or lactate dehydrogenase (ldhA) (EC 1.1.1.28, locus YP_(—)170916) are downregulated or knocked-out.

Certain enzymes described above provide pathways to assimilate CO₂ into the 2-carbon acetyl-CoA (reductive TCA and Woods-Ljungdahl pathways) or glyoxylate (3-HPA pathway). Combinations of these (preferentially the 3-HPA cycle and the reductive TCA cycle) are also engineered in special cases. In this scenario, the outputs of the CO₂ fixation reactions (acetyl-CoA and glyoxylate) are utilized as inputs for the glyoxylate cycle, which combines acetyl-CoA and glyoxylate into 4-carbon oxaloacetate (via a 4-carbon malate intermediate) [Chung T, Klumpp D J, Laporte D C. J Bacteriol (1988). “Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map.” 170(1):386-92.] Three key enzymes are involved in the glyoxylate shunt pathway. In preferred embodiments, all are overexpressed to maximize CO₂ fixation.

Malate synthase (EC 2.3.3.9) generates malate and coenzyme A from acetyl-CoA, water, and glyoxylate. An exemplary enzyme is encoded by E. coli locus JW3974 (aceB). Another exemplary activity is provided by an alternate malate synthase enzyme E. coli encodes, the JW2943 locus malate synthase G (glcB).

Isocitrate lyase (EC 4.1.3.1) generates glyoxylate and succinate from isocitrate. An exemplary enzyme is that encoded by E. coli locus JW3975 (aceA).

Malate dehydrogenase (EC 1.1.1.37) generates oxaloacetate and NADH from malate and NAD⁺. An exemplary enzyme is that encoded by E. coli locus JW3205 (mdh).

Gluconeogenesis is the process by which organisms generate glucose from non-sugar carbon substrates, including pyruvate, lactate, glycerol, and glucogenic amino acids. Most steps of glycolysis are bidirectional, with three exceptions (reviewed in Hers H G, Hue, L. Ann Rev. Biochem (1983). “Gluconeogenesis and related aspects of glycolysis.” 52:617-53). In certain embodiments, these enzyme activities are expressed to improve gluconeogenesis rates.

I. Conversion of Pyruvate to Phosphoenolpyruvate

Conversion of pyruvate to phosphoenolpyruvate requires two enzymatic activities as follows.

Pyruvate carboxylase (EC 6.4.4.1) generates oxaloacetate, ADP, and Pi from pyruvate, ATP, and CO₂. An exemplary pyruvate carboxylase is encoded by the YGL062W locus from Saccharomyces cerevisiae, pyc1.

Phosphoenolpyruvate carboxykinase (EC 4.1.1.49) generates phosphoenolpyurate, ADP, Pi, and CO₂ from oxaloacetate and ATP. An exemplary phosphoenolpyruvate carboxykinase is encoded by E. coli locus JW3366, pckA.

II. Conversion of Fructose 1,6-Bisphosphate to Fructose-6-Phosphate

Conversion of fructose 1,6-bisphosphate to fructose-6-phosphate requires fructose-1,6-bisphosphatase (EC 3.1.3.11), which generates fructose-6-phosphate and Pi from fructose-1,6-bisphosphate and water. An exemplary fructose-1,6-bisphosphatase is encoded by E. coli locus JW4191, fbp, (EC #4.1.2.13).

The Thermosynechoccocus elongates BP-1 genes fbpI (Accession Number: NP_(—)682066), fbpII (Accession Number: NP_(—)681331) and fbaA (Accession Number: NP_(—)681166) encoding, respectively, a fructose bisphosphatase I protein (E.C. 1.6.1.1), a fructose bisphosphatase II protein (E.C. 1.6.1.1) and a fructose bisphosphatase aldolase protein (E.C. XXX) were amplified directly from Thermosynechoccocus elongates BP-1 strain genomic DNA using the following primers: fbpI forward primer 5′-GAATACATATGACTGACTATGCAGCC-3′ (SEQ ID NO: 7) and reverse fbpI primer 5′-GAATAGAATTCTTACACCTGTGTCACGG-3′ (SEQ ID NO: 8); fbpII forward primer 5′-GAATAGTCTCATATGGATAACGTCATCGG-3′ (SEQ ID NO: 9) and reverse primer 5′-GAATAGAATTCTTAGTACAGCTGGAGTG-3′ (SEQ ID NO: 10); fbaA forward primer 5′-GAATACATATGGCACTCGTACCCATG-3′ (SEQ ID NO: 11) and reverse primer 5′-GAATAGAATTCTATAACCACCACGG-3′ (SEQ ID NO: 12). PCR amplifications were performed with PCR SuperMix High Fidelity (Invitrogen, Carlsbad, Calif.) and standard PCR amplification conditions. The fbpI and fbpII forward primer adds an NdeI restriction recognition site, the fbaA forward primer adds a BsmAI restriction recognition site, and all reverse primers add a stop codon and an EcoRI restriction recognition site.

The amplified fbpI, fbpII and fbaA PCR gene products were cloned into the pJB5 expression vector (“pJB5-fbpI;” “pJB5-fbpII;” “pJB5-fbaA”) by digesting the insert and vector individually with NdeI and EcoRI or BsmAI and EcoRI (New England Biolabs) restriction endonucleases with well known laboratory techniques. Digestions were gel isolated on 0.8% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen) and ligated with the T4 DNA Ligase (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into NEB 5-alpha chemically competent E. coli cells using standard techniques (New England Biolabs), and confirmed by PCR.

pJB5-fbpI, pJB5-fbpII and pJB5-fbaA plasmid stocks were purified using Qiagen miniprep kit for individual transformation into separate preparations of JCC136. One to two micrograms of the aforementioned plasmid DNAs were added to individual JCC136 cell preparations having an OD_(730nm) of 1 and incubated at 37 C for 4 hours using low intensity orbital shaking and low level light source. Cells were then plated onto A⁺ solid media plates and placed in a lighted incubator (100-250 μE/m2/s, 37 C) for 1 day. Twenty-five micrograms/ml_([final]) spectinomycin was underlayed on the plates and incubated until colonies grew (˜5 days). Integration into target Synechococcus host cells was confirmed by PCR of whole cell genomic DNA by a “colony PCR” protocol for the pJB5-fbpI transformation. Briefly, ˜1 mm colonies were resuspended in 50 μl deionized water, and 5 μl were used in 20 μl standard PCR reactions using Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.) with the addition of a 2 minute 98 C denaturation step at the very start of the standard PCR cycling conditions. The PCR showed correct bands for colonies, and the strain was named JCC136-FbpI (FIG. 10, panel B, lane 2).

A single colony of both JCC136 and JCC136-FbpI was grown in 10 mL A+ with 100 μg/ml_([final]) spectinomycin in a test tube immersed in a 37 C bath with bubbled 1% CO2. Cultures were grown to OD_(730nm) 5.0 or higher (Molecular Devices Spectramax M2e; previously determined that an OD_(730nm) of 1 is equal to ˜0.3 g CDW), and then spun down (21,000 RCF, 20 C, 5 min), resuspended in fresh A+ media to original concentration, and then appropriately back-diluted to OD_(730nm) 0.2 in 25 mL A⁺ in a baffled 125 mL shaker flask. Approximately 1 mL of culture was taken for each time point (0, 24, 48, 72, and 96 hours post-dilution, OD_(730nm) was recorded (appropriately diluted to give reading between 0.04 and 0.4, which was previously determined to be most accurate range on the Spectramax M2e). Samples were immediately spun down at 4 C for 10 min at 21,000 RCF. Supernatants were placed in a new tube, and frozen at −80 C until ready for analysis.

The supernatant from each time point was analyzed for ethanol and acetaldehyde by use of an Agilent 7890 Gas Chromatograph equipped with a headspace analyzer and a flame ionization detector (Agilent) using a J&W Scientific DB-ALC1 (Catalog Number: 123-9134; length: 30 m, Inner Diamter, 0.320 mm, Film Thickness: 1.80 μm). One hundred μl of each sample was subjected to headspace analysis. Controls were measured for A+ alone, and as well as from serial dilution of standards for ethanol and acetaldehyde obtained from Sigma to obtain a calibration curve. The levels of ethanol, and ethanol normalized with respect to the optical density are plotted in FIG. 11.

III. Conversion of Glucose-6-Phosphate to Glucose

Conversion of glucose-6-phosphate to glucose requires glucose-6-phosphatase (EC 3.1.3.68), which generates glucose and Pi from glucose-6-phosphate and water. An exemplary glucose-6-phosphatase is encoded by the Saccharomyces cerevisiae YHR044C locus, dog1. Another exemplary glucose-6-phosphatase activity is encoded by Saccharomyces cerevisiae YHR043C locus, dog2.

Oxaloacetate, the starting material for gluconeogenesis, is generated either via the glyoxylate shunt (leveraging inputs from the reductive TCA or Woods-Ljungdahl pathways and the 3-HPA pathway) or via the carboxylation of pyruvate. In the absence of the glyoxylate shunt, the pyruvate synthase activity of pyruvate ferredoxin:oxidoreductase (EC 1.2.7.1) can generate pyruvate, CoA, and oxidized ferredoxin from acetyl-CoA, CO₂, and reduced ferredoxin [Furdui C and Ragsdale S W. J. Biol. Chem (2000). “The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Woods-Ljungdahl pathway.” 275(37): 28494-99]. An exemplary pyruvate ferredoxin oxidoreductase with pyruvate synthase activity is encoded by locus Moth_(—)0064 from Moorella thermoaceticum.

Example 3 Engineering Reducing Power

The above CO₂-fixation pathways may require generating reducing power, primarily in the form of NADH (nicotinamide adenine dinucleotide, reduced form) and NADPH (nicotinamide adenine dinucleotide phosphate, reduced form).

Maintaining an appropriately-balanced supply of reduced NAD⁺ (NADH) and NADP⁺ (NADPH) is important to maximize carbon assimilation, and thus growth rate, of engineered photoautotrophic organisms.

Table 1 lists candidate genes for overexpression in the reducing power module together with information on associated pathways, Enzyme Commission (EC) Numbers, exemplary gene names, source organism, GenBank accession numbers, and homologs from alternate sources.

I. NADH

When NADH levels remain suboptimal, a plurality of methods is employed to increase intracellular NADH concentrations, including overexpression of the following genes.

NAD⁺-dependent isocitrate dehydrogenase (EC 1.1.1.41) generates 2-oxoglutarate, CO₂, and NADH from isocitrate and NAD⁺. Of note, most bacterial isocitrate dehydrogenases are NADP⁺-dependent (EC 1.1.1.42). An exemplary NAD⁺-dependent isocitrate dehydrogenase is the octameric Saccharomyces cerevisiae enzyme comprising locus YNL037C, idh1 and locus YOR136W, idh2.

Malate dehydrogenase (EC 1.1.1.37) generates oxaloacetate and NADH from malate and NAD⁺. Overexpression of NAD-dependent malate dehydrogenase can be employed to increase NADH pools. An exemplary enzyme is encoded by E. coli locus JW3205 (mdh).

The NADH:ubiquinone oxidoreductase from Rhodobacter capsulatus, is unique in its ability to reverse electron flow between the quinone pool and NAD⁺ [Dupuis A, Peinnequin A, Darrouzet E, Lunardi J. FEMS Microbiol Lett (1997). “Genetic disruption of the respiratory NADH-ubiquinone reductase of Rhodobacter capsulatus leads to an unexpected photosynthesis-negative phenotype.” 149:107-114; Dupuis A, Darrouzet E, Duborjal H, Pierrard B, Chevallet M, van Belzen R, Albracht S P J, Lunardi J. Mol. Microbiol (1998). “Distal genes of the nuo_operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH-ubiquinone oxidoreductase. 28:531-541]. The Rhodobacter Nuo operon, encoding the Nuo Complex I, can be reconstituted to generate additional NADH by reverse electron flow.

The Rhodobacter capsulatus nuo operon, locus AF029365, consisting of the 14 nuo genes nuoA-N (and 7 ORFs of unknown function) can be expressed to enable reverse electron flow and NADH-generation in photoautotrophic cells. The operon encodes NuoA, accession AAC24985.1; NuoB, accession AAC24986.1; NuoC, accession AAC24987.1; NuoD, accession AAC24988.1; NuoE, accession AAC24989.1; NuoF, accession AAC24991.1; NuoG, accession AAC24995.1; NuoH, accession AAC24997.1; NuoI, accession AAC24999.1; NuoJ, accession AAC25001.1; NuoK, accession AAC25002.1; NuoL, accession AAC25003.1; NuoM, accession AAC25004.1; and NuoN, accession AAC25005.1.

Expression of pyridine nucleotide transhydrogenase (EC 1.6.1.1) generates NADH and NADP⁺ from NADPH and NAD⁺. An exemplary enzyme is the E. coli soluble pyridine nucleotide transhydrogenase, encoded by sthA (also known as udhA), locus JW551. An alternate exemplary enzyme is the membrane bound E. coli pyridine nucleotide transhydrogenase, encoded by the multisubunit of NAD(P) transhydrogenase subunit alpha, encoded by pntA, locus JW1595, and NADP transhydrogenase subunit beta, encoded by pntB, locus JW1594.

Expression of udhA Gene Encoding Pyridine Nucleotide Transhydrogenase for NADH Production

The Escherichia coli K12 udhA gene (Accession Number: NP_(—)418397) encoding a pyridine nucleotide transhydrogenase (E.C.#1.6.1.1) was amplified directly from E. coli K12 strain genomic DNA using the forward primer 5′-TATGCCACATTCCTACGATTACGATGCC-3′ (SEQ ID NO: 13) and the reverse primer 5′-AATTCTTAAAACAGGCGGTTTAAACCGTTTAACGC-3′ (SEQ ID NO: 14). PCR amplifications were performed with the high fidelity Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.). The forward primer adds an NdeI restriction recognition site, and the reverse Primer adds a stop codon and an EcoRI restriction recognition site.

The amplified udhA PCR gene product was cloned into the pJB5 expression vector (“pJB5-udhA”) by digesting the insert and vector individually with NdeI and EcoRI (New England Biolabs) restriction endonucleases with well known laboratory techniques. Both digestions were gel isolated on 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 chemically competent cells using standard techniques (EpiCentre), and confirmed by PCR.

pJB5-udhA plasmid stocks were purified using Qiagen miniprep kit for transformation into Synechococcus sp. PCC 7002. One to two micrograms of pJB5-udhA plasmid was added to Synechococcus sp PCC 7002 cells grown to an optical density of 1 and incubated at 37 C for 4 hours using low intensity orbital shaking and low level light source. Cells were then plated onto A⁺ solid media plates and placed in a lighted incubator (100-250 μE/m2/s, 37 C) for 1 day. Twenty-five micrograms/mL spectinomycin was underplayed on the plates and incubated until colonies grew (˜5 days). Integration into target Synechococcus host cells is confirmed by PCR of whole cell genomic DNA by a “colony PCR” protocol. Briefly, ˜1 mm colonies were resuspended in 50 μl deionized water, and 5 μl were used in 20 μl standard PCR reactions using Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.) with the addition of a 2 minute 98 C degree denaturation step at the very start of the standard PCR cycling conditions. The PCR showed correct bands for colonies, and the strain was named JCC1-UdhA (FIG. 10, lane 2 and 3).

II. NADPH

NADPH serves as an electron donor in reductive (especially fatty acid) biosynthesis. Three parallel methods are used, singly or in combination, to maintain sufficient NADPH levels for photoautotrophy. Methods 1 and 2 are described in WO2001/007626, Methods for producing L-amino acids by increasing cellular NADPH. Method 3 is described in U.S. Pub. No. 2005/0196866, Increasing intracellular NADPH availability in E. coli.

Expression of the zwf Gene Encoding Glucose-6-Phosphate Dehydrogenase for NADPH

The Escherichia coli K12 zwf gene (Accession Number: AAC74922) encoding a glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was amplified directly from E. coli K12 strain genomic DNA using the forward primer 5′-TCGACATATGGCGGTAACGCAAACAGCCC-3′ (SEQ ID NO: 15) and the reverse primer 5′-TCGAGAATTCTTACTCAAACTCATTCCAGGAACGACCATC-3′ (SEQ ID NO: 16). PCR amplifications were performed with the high fidelity Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.). The forward primer adds an NdeI restriction recognition site, and the reverse Primer adds a stop codon and an EcoRI restriction recognition site.

The amplified zwf gene PCR product was cloned into the pJB5 expression vector (“pJB5-Zwf”) by digesting the insert and vector individually with NdeI and EcoRI (New England Biolabs) restriction endonucleases with well known laboratory techniques. Both digestions were gel isolated on 1% TAE agarose gel, purified using a Gel Isolation Kit (Qiagen) and ligated with the Quick Ligation Kit (New England Biolabs) with no deviation from the published techniques. The ligated product was transformed into EPI400 chemically competent cells using standard techniques (EpiCentre), and confirmed by PCR.

pJB5-Zwf plasmid stocks were purified using Qiagen miniprep kit for transformation into Synechococcus sp. PCC 7002. One to two micrograms of pJB5-Zwf plasmid was added to Synechococcus sp PCC 7002 cells grown to an optical density of 1 and incubated at 37 C for 4 hours using low intensity orbital shaking and low level light source. Cells were then plated onto A⁺ solid media plates and placed in a lighted incubator (100-250 μE/m2/s, 37 C) for 1 day. Twenty-five micrograms/ml spectinomycin was underplayed on the plates and incubated until colonies grew (˜5 days). Integration into target Synechococcus host cells is confirmed by PCR of whole cell genomic DNA by a “colony PCR” protocol. Briefly, ˜1 mm colonies were resuspended in 50 μl deionized water, and 5 μl were used in 20 μl standard PCR reactions using Phusion DNA Polymerase Master Mix (New England Biolabs, Beverly, Mass.) with the addition of a 2 minute 98 C degree denaturation step at the very start of the standard PCR cycling conditions. The PCR showed correct bands for colonies, and the strain was named JCC1-Zwf (FIG. 10, lane 4).

A. Increasing the Flux Through the Pentose Phosphate Pathway

Increasing the flux through the Pentose Phosphate Pathway generates 2 molecules of NADPH per molecule of glucose.

The downregulation or inactivation of the phosphoglucose isomerase, such as pgi from Synechococcus sp. PCC 6301 (locus YP_(—)172776), is known to force glucose through the pentose phosphate pathway. This therefore provides one approach for increasing intracellular NADPH pools, as has been previously applied in E. coli engineering [Kabir, M M. Shimizu, K. Appl. Microbiol. Biotechnol. (2003):Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production.” 62:244-255; Kabir M M, Shimizu K. J. Biotechnol (2003). “Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT-PCR” 105(1-2):11-31.]

Overexpression of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), which generates NADPH and 6-phospho-gluconolactone from glucose-6-phosphate and NADP⁺, provides another way to increase NADPH levels. An exemplary enzyme is that encoded by E. coli glucose-6-phosphate dehydrogenase, zwf, locus JW1841.

Overexpression of 6-phosphogluconolactonase (EC 3.1.1.31), which generates 6-phosphogluconate from 6-phosphoglucolactone and water, provides another approach for increasing flux through the pentose phosphate pathway. An exemplary enzyme is that encoded by the E. coli 6-phosphogluconolactonase, pgl, locus JW0750.

Overexpression of 6-phosphogluconate dehydrogenase (EC 1.1.1.44) generates ribose-5-phosphate, CO₂, and NADPH from 6-phosphogluconate and NADP⁺. This also can be used to increase NADPH levels by increasing flux through the pentose phosphate pathway. An exemplary enzyme is the encoded by E. coli 6-phosphogluconate dehydrogenase, gnd, locus JW2011.

B. Expression of NADP⁺-Dependent Enzymes

NADP⁺-dependent enzymes can be expressed in lieu of or in addition to NAD-dependent enzymes.

Overexpression of isocitrate dehydrogenase (EC 1.1.1.42) generates 2-oxoglutarate, CO₂, and NADPH from isocitrate and NADP⁺. An exemplary enzyme is encoded by the E. coli isocitrate dehydrogenase, icd, locus JW1122.

Overexpression of malic enzyme (EC 1.1.1.40) generates pyruvate, CO₂, and NADPH from malate and NADP⁺. An exemplary NADP-dependent enzyme is the E. coli malic enzyme, encoded by maeB, locus JW2447.

C. Expression of Pyridine Nucleotide Transhydrogenase

Expression of pyridine nucleotide transhydrogenase (EC 1.6.1.1) generates NADPH and NAD⁺ from NADH and NADP⁺. An exemplary enzyme is the E. coli soluble pyridine nucleotide transhydrogenase, encoded by sthA (also known as udhA), locus JW551. An alternate exemplary enzyme is the membrane bound E. coli pyridine nucleotide transhydrogenase, encoded by the multisubunit of NAD(P) transhydrogenase subunit alpha, encoded by pntA, locus JW1595 and NADP transhydrogenase subunit beta, encoded by pntB, locus JW1594.

Example 4 Improved Thermotolerance

In certain embodiments, photobioreactors are maintained at high operating temperatures. Engineering photoautotrophic organisms to withstand and thrive in high operating temperatures allows for reduced cooling costs and faster kinetics of carbon-based product output. Four significant ways to increase thermotolerance in photoautotrophic organisms include stabilizing the photosynthetic apparatus with accessory proteins, expressing chaperones to stabilize other intracellular proteins in the cell, altering the lipid profiles of cell membranes, and expressing osmoprotectants, such as betaine.

Photosystem II is the most heat sensitive protein in the photosynthetic apparatus [Berry J, Bjorkman O. Plant Physiol (1980) “Photosynthetic response and adaptation to temperature in higher plants.” 31:491]. The damage to Photosystem II is primarily located within the oxygen-evolving components of the complex, and degradation of and replacement of D1 represents a key step in the repair of damaged Photosystems [Nixon P J, Barker M, Boehm M, de Vries R, Komenda J. “FtsH-mediated repair of the photosystem II complex in response to light stress.” J. Exp. Biol (2005). 56(410:357-63]. In one embodiment, three genes—psbO, psbU, and psbV—that are known to protect these oxygen-evolving components in Photosystem II of Synechocystis sp. PCC 6803 are overexpressed to aid in thermotolerance [Kimura A, Eaton-Rye J J, Morita E H, Nishiyama Y, Hayashi H. Plant Cell Physiol. (2002). “Protection of the Oxygen-Evolving Machinery by the Extrinsic Proteins of Photosystem II is Essential for Development of Cellular Thermotolerance in Synechocystis sp. PCC 6803.” Plant Cell Physiol. 43(8):932-938]. These three exemplary genes encode amino acid sequences are Photosystem II manganese-stabilizing polypeptide (PsbO), locus NP_(—)441796; PS II complex 12 kDa extrinsic protein (PsbU), locus NP_(—)440167; and Cytochrome c550 (PsbV), locus NP_(—)441834.

A secondary source of thermal toxicity results from general protein instability at high temperatures. Several chaperones specifically confer thermotolerance by assisting protein folding and degradation at high temperatures including as ClpB [Eriksson M, Schelin J, Miskiewicz E, Clarke A K. J Bacteriol (2001) “Novel Form of ClpB/HSP100 Protein in the Cyanobacterium Synechococcus.” 183(24):7392-7396], GroESL [Rajaram H, Ballal A D, Apte S K, Wiegert T, Schumann W. Biochimica et Biophysica Acta (2001) “Cloning and characterization of the major groESL operon from a nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.” 1519(1-2):143-146], and HspA [Roy S K, Hiyama T, Nakamoto H. Eur J Biochem (1999). “Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an alpha-crystallin-related, small heat shock protein.” 262(2):406-416]. In the preferred embodiment, the orthologues of these genes from the thermophile Thermosynechococcus elongatus BP-1 are overexpressed to confer increased thermotolerance to the photoautotrophic organisms. These exemplary genes encode amino acid sequences are 16.6 kDa small heat shock protein molecular chaperone (HspA), locus NP_(—)681663; 60 kD chaperonin 1 (GroEL-1), locus NP_(—)680976; 60 kD chaperonin 2 (GroEL-2), locus NP_(—)682202; Co-chaperonin (GroES), locus NP_(—)680977; and Endopeptidase (ClpB), locus NP_(—)683242.

As an alternative or in addition, endogenous repressors of proteins involved in thermotolerance are downregulated or deleted. For example, exemplary regulatory genes that can be disrupted include hrcA, such as locus NP_(—)440130 of Synechocystis sp. PCC 6803 [Nakamoto H, Suzuki M, Kojima K. “Targeted inactivation of the hrcA repressor gene in cyanobacteria.” FEBS Lett (2003). 549(1-3):57-62] and the histidine kinase hik34, such as locus slr1285 of Synechocystis sp. PCC 6803, [Suzuki I, Kanesaki Y, Hayashi H, Hall J, Simon W J, Slabas A R, Murata N. “The histidine kinase Hik34 is involved in thermotolerance by regulating the expression of heat shock genes in Synechocystis.” Plant Physiol (2005). 138(3):1409-21.]

In addition or as an alternative, the lipid profile of cellular and intracellular membrane can be modified to increase thermotolerance. Of note, most thermotolerant organisms have evolved mechanisms to retain highly saturated fatty acids, frequently with lengthy and/or cyclic fatty acid tails. If natively expressed in the desired photoautotrophic organism, it is useful to downregulate or knock-out fatty acid desaturases (EC 1.14.19) such as delta-12-desaturase, for example the desA gene (locus NP_(—)441489) of Synechocystis sp. PCC 6803, delta-15 desaturase, for example the desB, locus NP_(—)441622 of Synechocystis sp. PCC 6803, stearoyl-CoA 9-desaturase (EC 1.14.19.1), for example the desC gene, locus NP_(—)442430 of Synechocystis sp. PCC 6803, and/or delta-6-desaturase (EC 1.14.19.3), for example the desD gene, locus NP_(—)441824 of Synechocystis sp. PCC 6803.

In addition or as an alternative, pathways to enable biosynthesis of the osmoprotectant betaine are engineered to improve thermotolerance of photoautotrophic organisms [Yang X, Wen X, Gong H, Lu Q, Yang Z, Tang Y, Liang Z, Lu C. “Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants.” Planta (2007). 225(3):719-33]. In this embodiment, a glycine sarcosine methyltransferase is expressed to convert glycine to sarcosine and sarcosine to dimethylglycine, as well as a dimethylglycine methyltransferase, to catalyze the methylation of dimethylglycine to betaine [Waditee R, Bhuiyan N H, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf A T, Takabe T, and Takabe T. “Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci (2005). 102(5):1318-23]. An exemplary glycine sarcosine methyltransferase gene is encoded by ApGSMT, locus BAC56939, from Aphanothece halophytica. An exemplary dimethylglycine methyltransferase is encoded by ApDMT, locus BAC56940 from Aphanothece halophytica.

Example 5 Improved pH Tolerance

In certain embodiments, elevated CO₂ levels are used to increase bioproductivity. However, elevating CO₂ levels in photobioreactors can cause concomitant increases to the acidity of the culture medium that can be toxic to the cell. Engineering cells to be tolerant to higher acidity conditions increases productivity and reduces costs associated with external pH control. In the present invention, one or more mechanisms to confer acid tolerance are engineered into the cell.

Two similar and complementary mechanisms of acid resistance use amino acid based decarboxylation and export to increase intracellular pH [Richard H, Foster J W. J Bacteriol (2004) “Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential.”186(18):6032-6041]. The genes required for the glutamate-based acid resistance are the glutamate decarboxylase isozymes GadA and GadB, as well as the glutamate/GABA antiporter GadC. The arginine-based acid resistance requires the arginine decarboxylase AdiA and the arginine/agmatine anti-porter AdiC. In addition, both acid-tolerance mechanisms require at least one of the Cl⁻ channels EriC and MriT [Iyer R, Iverson T M, Accardi A, Miller C. Nature (2002) “A biological role for prokaryotic ClC chloride channels.” 419(6908):715-718]. In the preferred embodiment, the genes required for one or both of these amino-acid based acid-resistance mechanisms from the enteric bacterium Escherichia coli K12 are overexpressed to confer increased acid tolerance. These exemplary genes encode amino acid sequences are glutamate decarboxylase A (GadA), EC 4.1.1.15, locus NP_(—)417974; Glutamate decarboxylase beta (GadB), 4.1.1.15, locus NP_(—)416010; glutamate: gamma-aminobutyric acid antiporter (GadC), locus NP_(—)416009; biodegradative arginine decarboxylase (AdiA), EC 4.1.1.19, locus NP_(—)418541); arginine:agmatin antiporter (AdiC), locus NP_(—)418539); Chloride channel protein (EriC), locus NP_(—)414697; and Chloride channel protein (MriT), locus NP_(—)416109.

In addition, or as an alternative, genes known to be involved in bacterial acid response mechanisms are overexpressed to confer acid tolerance to engineered bacteria. Thirty-two genes have been previously identified to be over-expressed in response to acid stress in response to acid stress [Ohta H, Shibata Y, Haseyama Y, Yoshino Y, Suzuki T, Kagasawa T, Kamei A, Ikeuchi M, Enami I. Photosynth Res (2005) “Identification of genes expressed in response to acid stress in Synechocystis sp. PCC 6803 using DNA microarrays.” 84(1-3):225-230]. In the preferred embodiment, the one or more of the genes expressed in the bacterium Synechocystis sp. PCC 6803 are overexpressed to confer increased acid tolerance. These exemplary genes encode amino acid sequences are Chaperone protein dnaK2 (DnaK), locus NP_(—)441989; DNA-directed RNA polymerase, sigma subunit (sll0306), locus NP_(—)441950; Zn-dependent protease (sll0528), locus NP_(—)442805; metal-dependent phosphoesterase (sll0549), locus NP_(—)442414; Acid-stress tolerance protein (sll0846), locus NP_(—)441124; Acid-stress related membrane protein (sll0939), locus NP_(—)440194; Acid-stress tolerance protein (sll1086), locus NP_(—)441667; Acid-stress tolerance protein (sll1483), locus NP_(—)442911; 16.6 kDa small heat shock protein, molecular chaperone (sll1514), locus NP_(—)440316; mannose-1-phosphate guanyltransferase (sll1558), EC 2.7.7.13, locus NP_(—)441699; RNA polymerase sigma factor (sll2012), locus NP_(—)441031; carboxyl-terminal processing protease (slr0008), EC 3.4.21.102, locus NP_(—)442119; molecular chaperone (slr0093), locus NP_(—)442496; Acid-stress tolerance protein (slr0270), locus NP_(—)441273; Geranylgeranyl pyrophosphate synthase (slr0611), locus NP_(—)439899; Acid-stress tolerance protein (slr0967), locus NP_(—)440193; CheY-like receiver (slr1214), locus NP_(—)440716; Signal transduction histidine kinase (slr1285), locus NP_(—)441610; Acid-stress tolerance protein (slr1413), locus NP_(—)440062; superoxide dismutase (slr1516), EC 1.15.1.1, locus NP_(—)441347; Acid-stress tolerance protein (slr1544), locus NP_(—)440790; Acid-stress tolerance protein (slr1573), locus NP_(—)442902; Acid-stress tolerance protein (slr1674), locus NP_(—)441676; hydrogenase expression/formation protein (slr1675), locus NP_(—)441677; Acid-stress tolerance protein (slr1676), locus NP_(—)441678; Acid-stress tolerance protein (slr1687), locus NP_(—)441698; Acid-stress tolerance protein (slr1915), locus NP_(—)440459; Esterase (slr1916), locus NP_(—)440460; Hydrogenase component protein (ssl3044), locus NP_(—)441697; Acid-stress tolerance protein (ssl3769), locus NP_(—)441305; Acid-stress tolerance protein (ssr2016), locus NP_(—)440709; Acid-stress tolerance protein (ssr2595), locus NP_(—)440789).

Example 6 Flue Gas Tolerance

Flue gas typically consists of N₂ (80%), CO₂(10-15%), O₂ (2-3%), as well as trace amounts of CO (70-110 ppm), NO_(x) (most typically NO₂) (50-70 ppm), SO₂ (180-250 ppm). Of the components of flue gas, only NO_(x) and SO₂ are thought to adversely effect the growth of photoautotrophic organisms [Negoro M, Shioji N, Miyamoto K, Miura Y. “Growth of microalgae in high CO₂ gas and effects of SO_(x) and NO_(x).” Appl Biochem Biotechnol (1991). Spring (28-29): 877-86.].

In preferred embodiments, photoautotrophic cells are engineered to exhibit improved tolerance to NO_(x), including NO₂. One exemplary means for improving tolerance to NO₂ is via overexpression of the ncgA (NP_(—)841001), ncgB (NP_(—)841000), and ncgC (NP_(—)840999) genes from Nitrosomonas europaea, [Beaumont H J, Lens S I, Westerhoff H V, and van Spanning R J. “Novel nirK cluster genes in Nitrosomonas europaea are required for NirK-dependent tolerance to nitrite.” J. Bacteriol (2005). 187(19):6849-51].

In preferred embodiments, photoautotrophic cells are engineered to exhibit improved tolerance to SO_(x), including SO₂. One exemplary means for improving tolerance to SO₂ is via overexpression of cysteine synthase A, cysK, from Synechococcus PCC 7942 (YP_(—)398721) [O-acetyl-L-Ser(thiol)-lyase] (EC 4.2.99.8 and 2.5.1.47) [Noji M, Saito, M, Nakamura M, Aono M, Saji H, Saito K. “Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants.” Plant Physiology (2001). 126:973-980]. In addition or as an alternative, superoxide dismutase is overexpressed, such as the exemplary sodA (NP_(—)441347; EC 1.15.1.1) gene from Synechocystis sp. PCC 6803. In preferred embodiments, catalase is also overexpressed, such as the katG (NP_(—)441295; EC 1.11.16) gene from Synechocystis sp. PCC 6803 [Tseng M J, Liu C W, Yiu J C. “Enhanced tolerance to sulfur dioxide and stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts.” Plant Physiol. Biochem (2007). 45(10-11):822-33].

Heavy metals such as lead, chromium, copper mercury and the like are byproducts of industrial combustive processes, commonly released through exhaust flue gases and pose a toxic threat to cyanobacteria. Genes have been identified that can be engineered into cyanobacteria to confer resistance to these and other heavy metals pollutants. For example, genes potentially relevant for imparting resistance to heavy metals can be found on natural plasmids pMOL28 (accession number NC_(—)006525) and pMOL30 (accession number NC_(—)006466). These plasmids were originally identified in Ralstonia metallidurans, a bacterium colonizing soils in industrial sediments, soils and wastes or in other regions having high levels of heavy metal toxicity. The pMOL28 and pMOL30 plasmids confer heavy metal resistance for cobalt, nickel and chromate (pMOL28) and cobalt, zinc, cadmium, copper, lead and mercury (pMOL30). For example, pMOL28 genes and proteins known to be involved with lead tolerance include pbrT (accession number YP_(—)145624), pbrR (accession number YP_(—)145623), Pb-efflux ATPase (accession number YO_(—)145622) and pbrD (accession number YP_(—)145620). pMOL28 genes involved in nickel and cobalt resistance include cnrC (accession number CAI30229), cnrA (accession number CAI30227), putative nickel and cobalt resistance genes (accession number CAI11305; CAI11304; CAI11303). Multi-substrate recognition proteins (cobalt-zinc-cadmium) are encoded by genes czcD (accession number YP_(—)145593.1), czcC (accession number YP_(—)145593.1) and czcB (accession number YP_(—)145594.1). pMOL30 genes for copper resistance include copA (accession number YP_(—)145682.1), copK (accession number Q58AD3; 2K0Q_A), copC (accession number YP_(—)145680.1), copD (accession number YP_(—)145679.1) and copper resistance transmembrane protein (accession number YP_(—)14682). pMOL30 genes for chromate resistance genes include chrB (accession number YP_(—)16177) and a gene for chromate transport protein (accession number YP_(—)161712). Other heavy metal resistance genes on pMOL30 can confer tolerance to mercury and include a gene for mercuric transport protein (accession number YP_(—)161729), a gene for periplasmic mercuric ion-binding protein (accession number YP_(—)161728), a gene for putative mercuric reductase (accession number YP_(—)161727) and a gene for putative mercury resistance protein (accession number YP_(—)161725).

Other sources of genes conferring metal resistance are identified in Sulfolobus solfataricus, comprising two genes for mercury resistance, merI (accession number ABL96631) and merH (accession number ABL96629) and a gene for mercuric reductase, merA (accession number ABL96630). Alternatively, an operon conferring mercury resistance has been identified in Streptomyces sp. CHR28, comprising mercuric reductase merA (accession number AAF64138), organomercurial lyase merB (accession number AAF64140), mercury transport merT (accession number AAF64136), orfIV (accession number AAF64134) and extracellular mercury binding protein merP (accession number AAF64135).

These examples represent only some of the potential genetic sources for conferring heavy metal resistance and are not to be construed as inclusive and limiting. For example, recently a Turkish group reported a plasmid mediated multiple heavy metal resistance from bacteria isolated from a landfill (M N Unaldi-Coral, et al., Annals Microbio. (2005) vol. 55(3):175-179). Other sources of metal resistance genes include plants and yeasts (S. Clemens, et al., EMBO (1999) vol. 18:3325-3333), Pseudomonas species (D. Reneiro, et al., Gene (1995) vol. 166:77-82) bacterial species such as Escherichia coli subjected to adaptive pressures from toxic environments (K R Brocklehurst and A P Morby, Microbiology (2000) vol. 146:2277-2282) and others.

Feasibility studies have shown that bacterial species can successfully be transformed and express genes mediating heavy metal tolerance. For example Escherichia species have been altered to confer mercury tolerance from genes found in other bacteria such as Bacillus (Y. Wang, et al., J. Bacteriology (1987) vol. 169:4848-4851) as well as higher organisms including mice (C C Huang, et al. Gene (1999) vol. 239:361-366.

Example 7 Engineered Salt Tolerance

In many geographic locales, available water supplies are affected by high salinity. As a result, in certain embodiments it is advantageous to propagate photoautotrophic organisms in brackish (8-500 mM NaCl) or sea water (˜530 mM). When the photoautotrophic cell of interest is unable to thrive under these conditions, it is necessary to overexpress nucleic acids conferring salt tolerance.

In one embodiment, a Na⁺/H⁺ antiporter is overexpressed. An exemplary Na⁺/H⁺ antiporter is the apnhaP gene (locus BAB69459) from the halotolerant cyanobacteria Aphanothece halophytica [Waditee R, Hibino T, Nakamura T, Incharoensakdi A, and Takabe T. “Overexpression of a Na⁺/H⁺ antiporter confers salt tolerance on a freshwater cyanobacterium, making it capable of growth in sea water.” Proc Natl Acad Sci (2002). 99(6):4109-4114]. In some instances, catalase is also overexpressed, such as the katG (NP_(—)441295; EC 1.11.16) gene from Synechocystis sp. PCC 6803 to improve growth rates in sea water.

In addition or as an alternative, the novel salt and cadmium stress related gene, scsr, locus BAE53693 of Chlamydomonas sp. W80 is expressed. [Tanaka S, Suda Y, Ikeda K, Ono M, Miyasaka H, Watanabe M, Sasaki K, and Hirata K. “A novel gene with antisalt and anticadmium stress activities from the halotolerant marine green alga Chlamydomonas sp. W80.” FEMS Microbiol Lett (2007). 271:48-52].

In addition or as an alternative the breast basic conserved gene, bbc1, locus BAA23724 of Chlamydomonas sp. W80 is expressed. [Tanaka S, Ikeda K, and Miyasaka H. “Enhanced tolerance against salt-stress and freezing-stress of Escherichia coli cells expressing algal bbc1 gene.” Curr. Microbiol (2001). 42:173-177].

In addition or as an alternative, pathways to enable biosynthesis of the osmoprotectant betaine are engineered to improve salt tolerance properties of photoautotrophic organisms. In this embodiment, a glycine sarcosine methyltransferase is expressed to convert glycine to sarcosine and sarcosine to dimethylglycine, as well as a dimethylglycine methyltransferase, to catalyze the methylation of dimethylglycine to betaine [Waditee R, Bhuiyan N H, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf A T, Takabe T, and Takabe T. “Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci (2005). 102(5):1318-23]. An exemplary glycine sarcosine methyltransferase gene is encoded by ApGSMT, locus BAC56839, from Aphanothece halophytica. An exemplary dimethylglycine methyltransferase is encoded by ApDMT, locus BAC56940 from Aphanothece halophytica.

Improved Salt-Tolerant Transgenic Cyanobacteria

A putative Na/H+ antiporter from Synechococcus sp. PCC 7002, which was identified by homology to the Na/H+ antiporter from Aphanothece halophytica (Accession Number: BAB69459), was amplified by PCR using the following primers:

NaH Forward Primer- (SEQ ID NO: 17) TCATCATATGCCTTTGGTCATGATTGTTTTAGCAGAAC, NaH Reverse Primer- (SEQ ID NO: 18) ATATCAATTGTTAACTCTGAATTGTTTTTTCGGTGGCTTTG. The Forward Primer adds an NdeI restriction endonuclease recognition site, and the Reverse Primer adds an MfeI restriction site.

The PCR product was cloned into pJB263 by digesting the insert and vector individually with NdeI and EcoRI (New England Biolabs). Both digestions were gel isolated on a 1% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen), and ligated using the Quick Ligation Kit (New England Biolabs) using standard techniques. The ligated product was transformed into EPI400 chemically competent E. coli cells using standard techniques (EpiCenter), and confirmed by PCR. The resulting plasmid, pJB263-NaH, was confirmed by PCR analysis.

Cells of Synechocystis sp. PCC 6803 are transformed with pJB263-NaH using the following procedure. pJB263-NaH plasmid stocks were purified using Qiagen miniprep kit for transformation into Synechocystis sp. PCC 6803. One to two micrograms of pJB263-NaH plasmid is added to Synechocystis sp PCC 6803 cells grown to an optical density of 1 and incubated at 30 C for 4 hours using low intensity orbital shaking and low level light source. Cells are then plated onto A⁺ solid media plates and is placed in a lighted incubator (100-250 uE/m2/s, 30 C) for 1 day. Twenty-five micrograms/mL spectinomycin is underlayed on the plates and incubated until colonies grew (˜5 days). Integration into target Synechocystis host cells is confirmed by PCR of whole cell genomic DNA by a “colony PCR” protocol:

Example 8 Nutrient Independence

In addition to CO2 and light, photoautotrophic organisms typically require inorganic nutrient sources and vitamins. Required nutrients are generally supplemented to the growth media during bench-scale propagation of such organisms. However, such nutrients are prohibitively expensive in the context of industrial scale bioprocessing.

Nitrogen is a key constituent of a variety of cellular macromolecules, including amino acids and nucleotides. Engineering photoautotrophs to efficiently utilize inexpensive sources provides significant economic and practical advantages.

In one embodiment, photoautotrophs are engineered to fix N₂, which is found present in concentrations of nearly 80% (v/v) in air and flue gas. In this embodiment, genes required for nitrogen fixation are overexpressed, in addition to genes required for synthesis of required cofactors and accessory protein [Herrero A, Muro-Pastor A M, Flores E. J Bacteriol (2001) “Nitrogen Control in Cyanobacteria”. 183(2): 411-425]. Eighteen such exemplary genes are found in Nostoc sp PCC 7120: FeMo cofactor biosynthesis protein (NifB), locus NP_(—)485557; [4Fe-4S] ferredoxin (FdxN), locus BAB77882; L-Cysteine desulfurase (NifS), EC 2.8.1.7, locus NP_(—)485499; Fe cluster accessory protein (NifU), locus NP_(—)485498; Nitrogenase-Fe subunit (NifH), EC 1.18.6.1, locus NP_(—)485497; Nitrogenase-alpha subunit (NifD), EC 1.18.6.1, locus NP_(—)485484; Nitrogenase-beta subunit (NifK), EC 1.18.6.1, locus NP_(—)485483; FeMo cofactor biosynthesis protein (NifE), locus NP_(—)485481; FeMo cofactor biosynthesis protein (NifN), locus NP_(—)485480; FeS cluster accessory protein (NifX), locus NP_(—)485479; FeMo cofactor accessory protein (NifW), locus NP_(—)485476; FeMo cofactor accessory protein (HesA), locus NP_(—)485475; FeS cofactor accessory protein (HesB), locus NP_(—)485474; Nitrogen-fixation specific ferredoxin (FdxH), locus NP_(—)485473; Pyruvate-flavodoxin oxidoreductase (NifJ), EC 1.2.7.1, locus NP_(—)486843; homocitrate synthase (NifV), EC 2.3.3.14, locus NP_(—)485450; FeMo cofactor accessory protein (NifZ), locus NP_(—)485451; and FeMo cofactor accessory protein (NifT), locus NP_(—)485452). Overexpression of the above genes enables photoautotrophic organisms to grow with N₂ gas as the sole source of nitrogen.

In addition or as an alternative, photoautotrophs are engineered to assimilate nitrite, which is a trace component of flue gas. In this embodiment, cells must be engineered to express a nitrite/nitrate transporter and be conveyed with the ability to convert nitrite into ammonia. Exemplary gene sequences encoding active nitrate/nitrate transporters are found Synechococcus sp. PCC 6301 [Herrero A, Muro-Pastor A M, Flores E. J Bacteriol (2001) “Nitrogen Control in Cyanobacteria”. 183(2): 411-425] and are overexpressed in cells to allow import of nitrates and nitrite, for example: ABC-type nitrate/nitrite transport system substrate-binding protein (NrtA), locus YP_(—)171021; ABC-type nitrate/nitrite transport system permease protein (NrtB), locus YP_(—)171022; ABC-type nitrate/nitrite transport system ATP-binding protein (NrtC), locus YP_(—)171023; ABC-type nitrate/nitrite transport system ATP-binding protein (NrtD), locus YP_(—)171024). As an alternative, the single polypeptide Nitrate/Nitrite transporter (NrtP) gene from Synechococcus sp. PCC 7002 is overexpressed [Sakamoto T, Inoue-Sakamoto K, Bryant D A. J Bacteriol (1999). “A Novel Nitrate/Nitrite Permease in the Marine Cyanobacterium Synechococcus sp. Strain PCC 7002.” 181(23):7363-7372], Nitrite/Nitrate permease (NrtP), locus AAD45941.

At elevated concentrations, nitrite is toxic to most cells. To alleviate nitrite toxicity, photoautotrophic cells are engineered to overexpress genes within the nitrite tolerance operon found in Nitrosomonas europaea ATCC 19718 [Beaumont H J E, Lens S I, Westerhoff H V, van Spanning R J M. “Novel nirK Cluster Genes in Nitrosomonas europaea Are Required for NirK-Dependent Tolerance to Nitrite.” J Bacteriol (2005). 187(19):6849-6851], Multicopper oxidase type 1 (NirK), locus NP_(—)840998; Cytochrome c, class IC (NcgA), locus NP_(—)841001; Cytochrome c, class I (NcgB), locus NP_(—)841000; Cytochrome c, class IC (NcgC), locus NP_(—)840999.

In addition or as an alternative, photoautotrophic cells are engineered to assimilate ammonia. In this embodiment, cells are engineered to overexpress an ammonium permease. An exemplary ammonium permease found in Synechocystis sp. PCC 6803 [Montesinos M L, Muro-Pastor A M, Herrero A, Flores E. “Ammonium/Methylammonium Permeases of a Cyanobacterium. Identification and analysis of three nitrogen-regulated amt genes in Synechocystis sp. PCC 6803.” J Biol Chem (1998). 273(47):31463-31470] is overexpressed High affinity ammonium/methylammonium permease (Amt1), locus NP_(—)442561; Ammonium/methylammonium permease (Amt2), locus NP_(—)440272; Ammonium/methylammonium permease (Amt3), locus NP_(—)442793.

In addition or as an alternative, photoautotrophic cells are engineered to assimilate urea. In this embodiment, cells must be engineered to overexpress a urea transporter to enable efficient uptake of urea into the cell. An exemplary urea transporter found in Nostoc sp. PCC 7120 [Valladares A, Montesinos M L, Herrero A, Flores E. “An ABC-type, high-affinity urea permease identified in cyanobacteria.” Molecular Microbiology (2002). 43(3):703-715] is overexpressed comprising the five gene ABC-type transporter for high affinity urea uptake for example ABC-type, high-affinity urea permease, periplasmic domain (UrtA), locus CAB70948.1; ABC-type, high-affinity urea permease, membrane domain (UrtB), locus CAB70949.1; ABC-type, high-affinity urea permease, membrane domain (UrtC), locus CAB70950.1; ABC-type, high-affinity urea permease, ATP binding domain (UrtD), locus CAB70951.1 and ABC-type, high-affinity urea permease, ATP binding domain (UrtE), locus CAB70952.1.

In addition, urea amidohydrolase (EC 3.5.1.5) (“urease”) and its associated accessory proteins are overexpressed, which catalyze conversion of urea to ammonia and carbon dioxide. An exemplary urease found in Synechococcus sp. WH 7805 [Collier J, Brahamsha B, Palenik B. “The marine cyanobacterium Synechococcus sp. WH7805 requires urease to utilize urea as a nitrogen source: molecular-genetic and biochemical analysis of the enzyme” Microbiology (1999). 145(2):447-459] is overexpressed comprising the ureABC urease genes: Urea amidohydrolase, gamma subunit (UreA), locus AAC61500; Urea amidohydrolase, beta subunit (UreB), locus AAC61501; Urea amidohydrolase, alpha subunit (UreC), locus AAC61502; and the ureDEFG genes encoding the accessory proteins Urease accessory protein (UreD), locus AAC61499; Urease accessory protein (UreE), locus AAC61498; Urease accessory protein (UreF), locus AAC61497; and Urease accessory protein (UreG), locus AAC61496.

Vitamin B12 is a vitamin cofactor that facilitates radical-based reaction catalyzation. Many organisms, including at least half of all microalgae surveyed, such as Synechococcus sp. PCC 7002, require external sources of Vitamin B12 for growth, which is prohibitively expensive in large-scale industrial bioprocessing [Croft M T, Warren M J, Smith A G. “Algae Need Their Vitamins”, Eukaryotic Cell (2006) 5(8):1175-1183]. In one embodiment, the need for Vitamin B12 is obviated by engineering photoautotrophic cells to express the Vitamin B12 biosynthesis pathway. An exemplary biosynthesis pathway found in Salmonella typhimurium is overexpressed, including but not limited to the following 20 genes: Uroporphyrin-III C-methyltransferase (CysG), EC 2.1.1.107, locus NP_(—)462380; Sirohydrochlorin cobaltochelatase (CbiK), EC 4.99.1.3, locus NP_(—)460970; Precorrin-2 C20-methyltransferase (CbiL), EC 2.1.1.130, locus NP_(—)460969; Precorrin-3B methylase (CbiH), EC 2.1.1.131, locus NP_(—)460972; Bifunctional CbiG/precorrin methyltransferase (CbiG), locus NP_(—)460973; Precorrin-4 C11-methyltransferase (CbiF), EC 2.1.1.133, locus NP_(—)460974; Cobalamin biosynthesis protein (CbiD), locus NP_(—)460977; NADPH-dependent precorrin-6A reductase (CbiJ), EC 1.3.1.54, locus NP_(—)460971; Precorrin-6B C5,15-methyltransferase (CbiE), EC 2.1.1.132, locus NP_(—)460976; Precorrin-6B C12 decarboxylase (CbiT), EC 2.1.1.132, locus NP_(—)460975; Precorrin-8X-methylmutase (CbiC), EC 5.4.1.2, locus NP_(—)460978; Cobyrinic acid A,C-diamide synthase (CbiA), EC 6.3.1.-, locus NP_(—)460980; Cob(I)yrinic acid a,c-diamide adenosyltransferase (BtuR), EC 2.5.1.17, locus NP_(—)460677; Cobyrinic acid synthase (CbiP), EC 6.3.5.10, locus NP_(—)460964; Cobyric acid decarboxylase (CobD), EC 4.1.1.81, locus NP_(—)459636; Adenosylcobinamide-phosphate synthase (CbiB), EC 6.3.1.10, locus NP_(—)460979; Alpha ribazole-5′-P phosphatase (CobC), EC 3.1.3.73, locus NP_(—)459635; Cobalamin(5′-phosphate) synthase (CobS), EC 2.7.8.26, locus NP_(—)460962; Cobinamide phosphate guanylyl transferase (CobU), EC 2.7.7.62, locus NP_(—)460963; and Nicotinate-nucleotide dimethylbenzimidazole-P phosphoribosyl transferase (CobT), EC 2.4.2.21, locus NP_(—)460961).

In addition, to allow for cobalt uptake and incorporation into Vitamin B12, the genes encoding the cobalt transporter are overexpressed. The exemplary cobalt transporter protein found in Salmonella typhimurium is overexpressed ABC-type Co2+ transport system, permease component (CbiM), locus NP_(—)460968; ABC-type cobalt transport system, periplasmic component (CbiN), locus NP_(—)460967; and ABC-type cobalt transport system, permease component (CbiQ), locus NP_(—)461989).

In a preferred embodiment, photoautotrophic organisms are engineered to overexpress Vitamin B12-independent enzymes to obviate the need for this cofactor entirely. In most photoautotrophic organisms, only methionine synthase (EC 2.1.1.13) and class II ribonucleotide reductases require Vitamin B12. An exemplary Vitamin B12-independent methionine synthase (EC 2.1.1.14) from Thermotoga maritima is therefore overexpressed: 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (MetE), locus NP_(—)229090 (SEQ ID NO: 22). In addition, an exemplary class I ribonucleotide reductase (nrdAB) from Synechocystis sp. PCC 6803 is overexpressed: Ribonucleoside-diphosphate reductase, alpha subunit (NrdA), locus NP_(—)441654; Ribonucleoside-diphosphate reductase, beta subunit (NrdB), locus NP_(—)443040.

It is furthermore contemplated that nutrient independence (e.g., Vitamin B12) of host cells of the present invention can be accomplished by expression of various proteins encoding 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (metE), ribonucleoside-diphosphate reductase, alpha subunit (nrdA), and ribonucleoside-diphosphate reductase, beta subunit (nrdB) comprising sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to metE, nrdA or nrdB.

Strain Construction

The expression plasmid, pJB5, contains two 500 bp regions of DNA homologous to sequences on the natural pAQ1 plasmid of Synechococcus sp. PCC 7002. Between the two regions of homology is a promoter and ribosome binding site, sites for DNA sequence insertion, and a resistance cassette, aadA1, that confers resistance to spectinomycin. The metE genes from E. coli (NP_(—)418273 (SEQ ID NO: 20)) and Thermosynechococcus elongatus BP-1 (NP_(—)681881 (SEQ ID NO: 21)) were encoded on pJB6 and pJB7 respectively. All the plasmids were digested with NdeI (New England Biolabs) and EcoRI (New England Biolabs), and the ˜1 kb fragment from pJB6 and pJB7, and the large fragment from pJB5, were gel isolated and purified using standard techniques (Qiagen). The fragments from pJB6 and pJB7 were ligated and transformed into pJB5 using standard techniques to form pJB5-6 and pJB5-7.

Synechococcus sp. PCC 7002 was transformed with pJB5-6 and pJB5-7 as follows. pJB5-6 and pJB5-7 were digested and inactivated with SbfI (New England Biolabs) for 1 hour and heat inactivated. DNA was incubated with fresh cells at OD730 of 1 for 4 hours in a dark incubator at 37° C. in A⁺. Cells were then allowed diluted in 20 mL fresh A⁺ media with moderate light and bubbled with air with 1% CO₂ for 24 hours. Cells were then diluted 1 in 20 into fresh A⁺ media containing 10 ug/mL spectinomycin and allowed to grow for 5 days under the same conditions. Cells were again diluted again 1 in 20 in 25 mL A⁺ media lacking Vitamin B12, and this was repeated a second time after the cells grew to high density. After the second outgrowth cells were plated onto A⁺ plates lacking Vitamin B12.

FIGS. 3A-D show wild-type Synechococcus sp. PCC 7002 and cells transgenically expressing E. coli MetE and Thermosynechococcus elongatus BP-1 MetE diluted and grown overnight in A⁺ media lacking Vitamin B12. Cells were diluted, then plated and allowed to grow 1 week at 37° C. in a lighted incubator on both Vitamin B12 sufficent and deficient plates. FIG. 3A illustrates wild-type Synechococcus on B12 sufficient plate whereas FIG. 3B shows B12 deficient plate. FIG. 3C represents a transgenic Synechococcus strain with the E. coli MetE on Vitamin B12 deficient plate and FIG. 3D shows a Thermosynechococcus elongatus MetE on Vitamin B12 deficient plate. The results show the ability of transgenically expressed methionine synthase to rescue the Vitamin B12 requirements of cyanobacteria.

Example 9 Near Infra Red Absorbance

Acaryochloris marina is the only known organism to have chlorophyll (Chl) d as its main Chl constituent. Chl d absorbs far-red/near it light in the range of 700 nm-750 nm and carries out oxygenic photosynthesis. It also contains Chl a, found in many organisms as the primary constituent. Chl a differs from Chl d only in that it has a vinyl group in place of a formyl group. We reasoned, then, that Chl d is derived from Chl a (or that a precursor of Chl d is derived from the analogous precursor of Chl a) by one or more of the mechanisms shown in FIG. 4.

The Acaryochloris marina genome has been sequenced and annotated, making it possible to locate putative oxygenase- and epoxide hydrolase-encoding genes contained within it. FIG. 6 lists those genes explicitly identified in the annotation of the A. marina as some form of “oxygenase”. Because these identifications were made by homology comparisons with other known oxygenases, a further BLAST search was not conducted. FIG. 7 lists the genes from A. marina with the most similarity to the Anabaena variabilis gene encoding EC 3.2.2.3 (epoxide hydrolase), determined by Protein BLAST. All genes with an expect value of less than 0.5 are shown. The protein sequence used as the query is shown in FIG. 5.

It is noted in Swingley et al., PNAS 105:2005 (2008) that an alternative method of oxygenation is that which transfers oxygen from water using S-adenosylmethionine (SAM). The genes encoding putative proteins of this type are given in FIG. 8.

The protein corresponding to locus tag AM1_(—)5665 (protein id ABW30612.1) [Acaryochloris marina MBIC11017] (FIG. 6B) was noted by Swingley et al., PNAS 105:2005 (2008) as being a likely oxygenase but not having significant homology with known examples. This could mean it is especially significant in Chl d formation.

The protein corresponding to locus tag AM1_(—)2935 (protein id ABW27932.1) [Acaryochloris marina MBIC11017], a likely thioredoxin, was found in the GenBank annotation as the only gene explicitly containing “epox” (see FIG. 7).

The protein corresponding to locus tag AM1_(—)5023) (protein id ABW29989.1) [Acaryochloris marina MBIC11017] and the protein corresponding to locus tag AM1_(—)5798) (protein id ABW30743.1) [Acaryochloris marina MBIC11017] share little homology with other sequenced cyanobacteria, according to Swingley et al., PNAS 105:2005 (2008) and thus may be important in Chl d synthesis.

The Acaryochloris marina genes and the encoded protein sequences for photosystem proteins that can use chlorophyll d as a photoreceptor are provided in Japanese Kokai No. 2001-346585, titled “Photosystem proteins that can use chlorophyll d as a photoreceptor and the genes that code them,” published Dec. 18, 2001, corresponding to Application number P2000-170696, filed June 7, 200 by Marine Biotechnology Institute Co., Ltd, 1-28-10 Hongo, Bunkyo-ku, Tokyo, the entire disclosure of which is hereby incorporated by reference in its entirety. Amino acid sequences of six photosystem proteins corresponding to Acaryochloris marina locus tags AM1_(—)2457, AM1_(—)2458, AM1_(—)2166, AM1_(—)1083, AM1_(—)2026, and AM1_(—)1084 were retrieved from the Japanese Kokai No. 2001-346585 application and used as query sequences in BLASTP searches. The BLASTP searches identified full-length amino acid sequences corresponding to the six photosystem proteins set out in Table 6, which appears in FIG. 9.

The protein corresponding to Acaryochloris marina locus tag AM1_(—)2457 (protein id ABW27465.1) [Acaryochloris marina MBIC11017] is the photosystem I core protein PsaA. The protein corresponding to Acaryochloris marina locus tag AM1_(—)2458 (protein id ABW27466.1) [Acaryochloris marina MBIC11017] is photosystem I core protein PsaB. Together, these two subunit proteins assemble to form the photosystem I protein that converts light energy into redox power and reduces NADP using electrons supplied by photosystem II.

The protein corresponding to Acaryochloris marina locus tag AM1_(—)2166) (protein id ABW27180.1) [Acaryochloris marina MBIC11017] is photosystem II D1 protein PsbA. The protein corresponding to Acaryochloris marina locus tag AM1_(—)1083) (protein id ABW26122.1) [Acaryochloris marina MBIC11017] is photosystem II D2 protein PsbD. Together PsbA and PsbD assemble and form the photosystem II protein. The photosystem II protein converts light energy into redox power, decomposes water and takes up electrons and supplies them to photosystem I.

The protein corresponding to Acaryochloris marina locus tag AM1_(—)2026 (protein id ABW27041.1) [Acaryochloris marina MBIC11017] is photosystem II CP47 protein PsbB. The protein corresponding to Acaryochloris marina locus tag AM1_(—)1084) (protein id ABW26123.1) [Acaryochloris marina MBIC11017] is photosystem II CP43 protein PsbC. PsbB and PsbC assemble and form the photosystem II core antenna proteins. Energy absorbed by the antenna to capture light energy used to decompose water is transmitted to the reaction center protein.

According to one embodiment of the invention a photoautotroph is created to absorb far-red/near IR light (i.e., in the neighborhood of 700 nm-750 nm). In this embodiment, genes required for synthesis of chlorophyll d and photosystem proteins I and II capable of binding chlorophyll d and using it as a photoreceptor for transducing light energy into reducing power are overexpressed. Exemplary genes include those encoding any of the Acaryochloris marina proteins disclosed herein.

TABLE 7 Informal Sequence Listing (5′→ 3′) (SEQ ID NOS 1-2 and 5-18) TTGCTACCTGCAGGGCCACCACAGCCAAATTCATCGTT GGTTGTGCGGCCGCAGTATTGGCTGTGATGTTGG TATACATCATATGGGTAAATTATTACTGATATTAGGTAGTGTTATTGC GCTACAATTGTTAAGCATTAGAAGATTCTTTAACAGCAACATTCC GAATACATATGACTGACTATGCAGCC GAATAGAATTCTTACACCTGTGTCACGG GAATAGTCTCATATGGATAACGTCATCGG GAATAGAATTCTTAGTACAGCTGGAGTG GAATACATATGGCACTCGTACCCATG GAATAGAATTCTATAACCACCACGG TATGCCACATTCCTACGATTACGATGCC AATTCTTAAAACAGGCGGTTTAAACCGTTTAACGC TCGACATATGGCGGTAACGCAAACAGCCC TCGAGAATTCTTACTCAAACTCATTCCAGGAACGACCATC TCATCATATGCCTTTGGTCATGATTGTTTTAGCAGAAC ATATCAATTGTTAACTCTGAATTGTTTTTTCGGTGGCTTTG

All references to publications, including scientific publications, treatises, pre-grant patent publications, and issued patents are hereby incorporated by reference in their entirety for all purposes. The teachings of the specification are intended to exemplify but not limit the invention, the scope of which is determined by the following claims. 

1.-55. (canceled)
 56. An engineered cyanobacterial cell for fuel production, wherein said cell comprises a recombinant nucleic acid encoding an enzyme with fructose-1,6-bisphosphatase activity.
 57. The engineered cyanobacterial cell of claim 56, wherein said enzyme is Thermosynechoccocus elongatus BP-1 fructose-1,6-bisphosphatase.
 58. The engineered cyanobacterial cell of claim 57, wherein said cyanobacterial cell is a Synechococcus species.
 59. The engineered cyanobacterial cell of claim 58, wherein said Synechococcus species is Synechococcus sp. PCC
 7002. 60. The engineered cyanobacterial cell of claim 56, wherein said cyanobacterial cell is a Synechococcus species.
 61. The engineered cyanobacterial cell of claim 60, wherein said Synechococcus species is Synechococcus sp. PCC
 7002. 62. A method for increasing flux through the Calvin cycle and carbon-fixation in a cyanobacterial cell, comprising transforming said cyanobacterial cell with a nucleic acid encoding an enzyme with fructose-1,6-bisphosphatase activity.
 63. The method of claim 62, wherein said enzyme is Thermosynechoccocus elongatus BP-1 fructose-1,6-bisphosphatase.
 64. The method of claim 63, wherein said cyanobacterial cell is a Synechococcus species.
 65. The method of claim 64, wherein said cyanobacterial cell is Synechococcus sp. PCC
 7002. 66. A method to produce a carbon-based product of interest, comprising culturing an engineered cyanobacterial cell in the presence of CO2 and light under conditions suitable to produce a carbon-based product of interest, wherein said engineered cyanobacterial cell comprises a recombinant nucleic acid encoding an enzyme with fructose-1,6-bisphosphatase activity.
 67. The method of claim 66, wherein said fructose-1,6-bisphosphatase is Thermosynechoccocus elongatus BP-1 fructose-1,6-bisphosphatase.
 68. The method of claim 66, wherein said cyanobacterial cell is a Synechococcus species.
 69. The method of claim 67, wherein said cyanobacterial cell is Synechococcus sp. PCC
 7002. 70. The method of claim 68, wherein said Synechococcus species is Synechococcus sp. PCC
 7002. 